Polypeptides Having Phospholipase C Activity and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to a method of reducing the phospholipid content in an oil or fat composition and polypeptides having PI-specific phospholipase C activity as well as polypeptides having PC, PE-specific phospholipase C activity and combinations thereof capable of catalyzing this reduction. The invention also relates to polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/423,748filed May 28, 2019, now pending, which is a divisional of U.S.application Ser. No. 15/126,098 filed on Sep. 14, 2016, now U.S. Pat.No. 10,351,795, which is a 35 U.S.C. 371 national application ofinternational application no. PCT/EP2015/055856 filed on Mar. 19, 2015,which claims priority or the benefit under 35 U.S.C. 119 of Europeanapplication no. 14160698.8 filed on Mar. 19, 2014. The content of eachapplication is fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a method of reducing the phospholipidcontent in an oil composition and polypeptides having phospholipase Cactivity capable of catalyzing this reduction. The invention alsorelates to polynucleotides encoding the polypeptides, nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing and using the polypeptides.

Description of the Related Art

Several types of phospholipases are known which differ in theirspecificity according to the position of the bond attacked in thephospholipid molecule. Phospholipase A1 (PLA1) removes the 1-positionfatty acid to produce free fatty acid and 1-lyso-2-acylphospholipid.Phospholipase A2 (PLA2) removes the 2-position fatty acid to producefree fatty acid and 1-acyl-2-lysophospholipid. The term phospholipase B(PLB) is used for phospholipases having both A1 and A2 activity.Phospholipase C (PLC) removes the phosphate moiety to produce 1,2diacylglycerol and phosphate ester. Phospholipase D (PLD) produces1,2-diacylglycero-phosphate and base group (See FIG. 1).

Before consumption vegetable oils are degummed to provide refinedstorage stable vegetable oils of neutral taste and light color. Thedegumming process comprises removing the phospholipid components (thegum) from the triglyceride rich oil fraction. The most commonly usedprocesses in the industry are water degumming, chemical/caustic refiningand physical refining including acid assisted degumming and/or enzymeassisted degumming. Due to the emulsifying properties of thephospholipid components, the degumming procedure has resulted in a lossof oil, i.e., of triglycerides.

Enzymatic degumming reduces the oils loss due to an efficient hydrolysisof the phospholipids which decrease the emulsifying properties. For areview on enzymatic degumming, see, Dijkstra, 2010, Eur. J. Lipid Sci.Technol. 112: 1178. The use of Phospholipase A and/or phospholipase C indegumming is for example described in Clausen, 2001, Eur. J. Lipid Sci.Technol. 103 333-340, WO 2003/089620 and WO 2008/094847. Phospholipase Asolutions generate lysophospholipid and free fatty acids resulting inoil loss. Phospholipase C on the other hand has the advantage that itproduces diglyceride (FIG. 2) which will remain in the oil and thereforewill reduce losses. There are four major phospholipids in vegetable oilphosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidicacid (PA) and phosphatidyl inositol (PI). Phospholipase C enzymes havedifferent specificity towards these phospholipids. The only knowncommercially available phospholipase C is Purifine of Verenium/DSM(Dijkstra, 101st AOCS Annual Meeting 10. May 2010) which has specificitytowards PC and PE. WO 2007/059927 describes a thermostable Bacillus PLCfor degumming. WO 2012/062817 describes a fungal PLC with specificitytowards all four phospholipids. A PI-specific phospholipase C has beendescribed in WO 2011/046815.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates where different phospholipases cleave a phospholipidas well as the four major functional groups on phospholipids.

FIG. 2 illustrates the reaction of a phospholipid with a phospholipase Cto form diglyceride and a phosphate ester or phosphoric acid.

SUMMARY OF THE INVENTION

The present invention provides a method for reducing the content ofphospholipids in an oil composition, the method comprising

a) providing an oil composition containing a quantity of phospholipids,

b) contacting said oil composition with a phosphatidylinositolphospholipase C and a PC and PE-specific phospholipase C underconditions sufficient for the enzymes to react with the phospholipids tocreate diglyceride and phosphate ester, and

c) separating the phosphate ester from the oil composition. Inparticular, the said phosphatidylinositol phospholipase C is from thegenus of Pseudomonas.

The invention further provides a polypeptide having phosphatidylinositolphospholipase C activity, selected from the group consisting of:

a) a polypeptide having at least 91% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

b) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions with

-   -   i) the mature polypeptide coding sequence of SEQ ID NO: 1, or    -   ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more positions; and

e) a fragment of the polypeptide of (a), (b), (c), or (d) that hasphosphatidylinositol phospholipase C activity.

In addition, the invention provides a polypeptide having PC and PEspecific phospholipase C activity, selected from the group consistingof:

a) a polypeptide having at least 70% sequence identity to the maturepolypeptide of SEQ ID NO: 19;

b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with

-   -   i) the mature polypeptide coding sequence of SEQ ID NO: 18, or    -   ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 70%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 18;

d) a variant of the mature polypeptide of SEQ ID NO: 19 comprising asubstitution, deletion, and/or insertion at one or more positions; and

e) a fragment of the polypeptide of (a), (b), (c), or (d) that has PCand PE specific phospholipase C activity.

Finally, the invention provides a composition comprising a mixture of aphosphatidylinositol phospholipase C from the genus of Pseudomonas and aPC and PE-specific phospholipase C polypeptide.

Definitions

Phospholipase C activity: The term “phospholipase C activity” or “PLCactivity” relates to an enzymatic activity that removes the phosphateester moiety from a phospholipid to produce a 1,2 diacylglycerol (seeFIG. 2). Most PLC enzymes belong to the family of hydrolases andphosphodiesterases and are generally classified as EC 3.1.4.3. Some PLCenzymes are classified in other EC classes, for example PI-specificPLC's. Phospholipase C activity may be determined according to theprocedure described in Example 5 or by one of the assays described inthe “Assay for phospholipase activity” section.

Phospholipase C specificity: The term “phospholipase C specificity”relate to a polypeptide having phospholipase C activity where theactivity is specified towards one or more phospholipids, with the fourmost important once being phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylinositol (PI) (see FIG. 1). Phospholipase C specificity may bedetermined by ³²P-NMR as described in Example 5.

PC and PE-specific phospholipase C: The term “PC and PE-specificphospholipase C” or “PC, PE-specific phospholipase C” relates to apolypeptide having activity towards phosphatidylcholine (PC),phosphatidylethanolamine (PE). In addition to the PC and PE specificityit may also have some activity towards phosphatidic acid (PA) andphosphatidyl inositol (PI). Preferably a PC and PE specificphospholipase C removes at least 30% PC and at least 30% PE from an oilor fat with at least 100 ppm PC and 100 ppm PE when using the P-NM Rassay of Example 5 at the optimal pH of the enzyme and an enzyme dosageof 10 mg/kg. More preferably it removes 40%, 50%, 60%, 70% or 80%, evenmore preferred it removes 90% and most preferred it removes between 90%and 100% of the PC in the oil or fat and 40%, 50%, 60%, 70% or 80%, evenmore preferred it removes 90% and most preferred it removes between 90%and 100% of the PE in the oil or fat.

PI-Specific Phospholipase C: The term “PI-specific phospholipase C” or“Phosphatidylinositol phospholipase C” relates to a polypeptide havingactivity towards phosphatidyl inositol (PI), meaning that its activitytowards phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidic acid (PA) is low compared to the PI activity. PI-specificphospholipase C enzymes can either belong to the family of hydrolasesand phosphodiesterases classified as EC 3.1.4.11 or to the family oflyases classified as EC 4.6.1.13. PI-specific phospholipase C activitymay be determined according to the procedure described in Example 5.Preferably a PI-specific phospholipase C removes at least 30% PI from anoil or fat with at least 50 ppm PI when using the P-NMR assay of Example5 at the optimal pH of the enzyme and an enzyme dosage of 10 mg/kg. Morepreferably it removes 40%, 50%, 60%, 70% or 80%, even more preferred itremoves 90% and most preferred it removes between 90% and 100% of the PIin the oil or fat.

Preferably a PI-specific Phospholipase C removes at least 20% more PIwhen compared to the amount of PC, PE or PA it can remove, morepreferred at least 30%, 40%, even more preferred at least 50% and mostpreferred at least 60% more PI when compared to the amount of PC, PE orPA it can remove.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Crude oil: The term “crude oil” refers to (also called a non-degummedoil) a pressed or extracted oil or a mixture thereof from, e.g.,vegetable sources, including but not limited to acai oil, almond oil,babassu oil, blackcurrent seed oil, borage seed oil, canola oil, cashewoil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil,crambe oil, flax seed oil, grape seed oil, hazelnut oil, hempseed oil,jatropha oil, jojoba oil, linseed oil, macadamia nut oil, mango kerneloil, meadowfoam oil, mustard oil, neat's foot oil, olive oil, palm oil,palm kernel oil, palm olein, peanut oil, pecan oil, pine nut oil,pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, saffloweroil, sasanqua oil, sesame oil, shea butter, soybean oil, sunflower seedoil, tall oil, tsubaki oil walnut oil, varieties of “natural” oilshaving altered fatty acid compositions via Genetically ModifiedOrganisms (GMO) or traditional “breading” such as high oleic, lowlinolenic, or low saturated oils (high oleic canola oil, low linolenicsoybean oil or high stearic sunflower oils).

Degummed oil: The term “degummed oil” refers to an oil obtained afterremoval of nonhydratable phospholipids, hydratable phospholipids, andlecithins (known collectively as “gums”) from the oil to produce adegummed oil or fat product that can be used for food production and/ornon-food applications, e.g., biodiesel. In certain embodiments, thedegummed oil has the phospholipids content of less than 200 ppmphosphorous, such as less than 150 ppm phosphorous, less than 100 ppmphosphorous, less than (or less than about) 50 ppm phosphorous, lessthan (or less than about) 40 ppm phosphorous, less than (or less thanabout) 30 ppm phosphorous, less than (or less than about) 20 ppmphosphorous, less than (or less than about) 15 ppm phosphorous, lessthan (or less than about) 10 ppm phosphorous, less than (or less thanabout) 7 ppm phosphorous, less than (or less than about) 5 ppmphosphorous, less than (or less than about) 3 ppm phosphorous or lessthan (or less than about) 1 ppm phosphorous.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide or domain; wherein the fragment hasphospholipase C activity. The fragments according to the invention havea size of more than approximately 200 amino acid residues, preferablymore than 210 amino acid residues, more preferred more than 220 aminoacid residues, more preferred more than 230 amino acid residues (e.g.,amino acids 44 to 278 or amino acid 34 to 268 of SEQ ID NO: 19), morepreferred more than 240 amino acid residues (e.g., amino acids 39 to 278or amino acid 34 to 273 of SEQ ID NO: 19), more preferred more than 250amino acid residues, more preferred more than 260 amino acid residues,more preferred more than 270 amino acid residues, and most preferredmore than 280 amino acid residues. In one aspect, a fragment contains atleast 290 amino acid residues (e.g., amino acids 33 to 322 or amino acid26 to 315 of SEQ ID NO: 2), at least 294 amino acid residues (e.g.,amino acids 29 to 322 or amino acid 26 to 319 of SEQ ID NO: 2), or atleast 296 amino acid residues (e.g., amino acids 27 to 322 or amino acid26 to 321 of SEQ ID NO: 2).

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 26 to 322 of SEQ ID NO: 2 based on the SignalP version 3 program (Nielsen et al., 1997, Protein Engineering 10: 1-6)which predicts that amino acids 1 to 25 of SEQ ID NO: 2 are a signalpeptide. When expressed in Bacillus as described in Example 1 anadditional alanine is added to the N-terminal. The N-terminal sequenceof the mature polypeptide (SEQ ID NO: 3) was confirmed to be AQESPAF(See Example 3). In another aspect the mature polypeptide is amino acids34 to 278 of SEQ ID NO: 19 based on the Signal P version 3 program(Nielsen et al., 1997, Protein Engineering 10: 1-6) which predicts thatamino acids 1 to 34 of SEQ ID NO: 19 are a signal peptide. Whenexpressed in Bacillus as described in Example 1 an additional alanine isadded to the N-terminal. The N-terminal sequence of the maturepolypeptide (SEQ ID NO: 20) was confirmed to be AWSADAP (See Example 3).It is known in the art that a host cell may produce a mixture of two ormore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide. Itis also known in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide. In one aspect, a maturepolypeptide of SEQ ID NO: 2 contains up to at least 300 amino acidresidues (e.g., amino acids 23 to 322 of SEQ ID NO: 2), or at least 299amino acid residues (e.g., amino acids 24 to 322 of SEQ ID NO: 2) or atleast 298 amino acid residues (e.g., amino acids 25 to 322 of SEQ ID NO:2) or at least 296 amino acid residues (e.g., amino acids 27 to 322 ofSEQ ID NO: 2) or at least 295 amino acid residues (e.g., amino acids 28to 322 of SEQ ID NO: 2). In another aspect, a mature polypeptide of SEQID NO: 19 contains up to at least 247 amino acid residues (e.g., aminoacids 31 to 278 of SEQ ID NO: 19), or at least 246 amino acid residues(e.g., amino acids 32 to 278 of SEQ ID NO: 19) or at least 244 aminoacid residues (e.g., amino acids 35 to 278 of SEQ ID NO: 19) or at least243 amino acid residues (e.g., amino acids 36 to 278 of SEQ ID NO: 19).

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving phospholipase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 76 to 966 of SEQ ID NO: 1 based onSignalP (Nielsen et al., 1997, supra)] that predicts nucleotides 1 to 75of SEQ ID NO: 1 encodes a signal peptide. In another aspect, the maturepolypeptide coding sequence is nucleotides 100 to 837 of SEQ ID NO: 18based on SignalP (Nielsen et al., 1997, supra)] that predictsnucleotides 1 to 99 of SEQ ID NO: 18 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Variant: The term “variant” means a polypeptide having phospholipase Cactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Nucleic Acid Sequences and Amino Acid Sequences

SEQ ID NO: 1: PI-specific PLC, Pseudomonas sp.; coding sequence.SEQ ID NO: 2: PI-specific PLC, Pseudomonas sp.; amino acid sequence.SEQ ID NO: 3: PI-specific PLC, Pseudomonas sp.; amino acid sequence ofmature polypeptide with N-terminal Alanine.SEQ ID NO: 4: PI-specific PLC, Pseudomonas chlorapsis; coding sequence.SEQ ID NO: 5: PI-specific PLC, Pseudomonas chlorapsis; amino acidsequence.SEQ ID NO: 6: PI-specific PLC, Pseudomonas chlorapsis; amino acidsequence of mature polypeptide with N-terminal Alanine.SEQ ID NO: 7: PI-specific PLC, Pseudomonas sp.; coding sequence.SEQ ID NO: 8: PI-specific PLC, Pseudomonas sp.; amino acid sequence.SEQ ID NO: 9: PI-specific PLC, Pseudomonas sp.; amino acid sequence ofmature polypeptide with N-terminal Alanine.SEQ ID NO: 10: PI-specific PLC, Pseudomonas chlorapsis; coding sequence.SEQ ID NO: 11: PI-specific PLC, Pseudomonas chlorapsis; amino acidsequence.SEQ ID NO: 12: PI-specific PLC, Pseudomonas chlorapsis; amino acidsequence of mature polypeptide with N-terminal Alanine.SEQ ID NO: 13: PI-specific PLC, Pseudomonas protegens; coding sequence.SEQ ID NO: 14: PI-specific PLC, Pseudomonas protegens; amino acidsequence.SEQ ID NO: 15: PI-specific PLC, Pseudomonas protegens; amino acidsequence of mature polypeptide with N-terminal Alanine.SEQ ID NO: 16: PI-specific PLC, Pseudomonas protegens; coding sequence.SEQ ID NO: 17: PI-specific PLC, Pseudomonas protegens; amino acidsequence.SEQ ID NO: 18: PC/PE-specific PLC, Bacillus sp. coding sequence.SEQ ID NO: 19: PC/PE-specific PLC, Bacillus sp. amino acid sequence.SEQ ID NO: 20: PC/PE-specific PLC, Bacillus sp. amino acid sequence ofmature polypeptide with N-terminal Alanine.SEQ ID NO: 21: Bt-PLC Bacillus thuringiensis; coding sequence.SEQ ID NO: 22: Bt-PLC Bacillus thuringiensis; amino acid sequence ofmature polypeptide.SEQ ID NO: 23: PC/PE-specific PLC, Bacillus pseudomycoides; codingsequence.SEQ ID NO: 24: PC/PE-specific PLC, Bacillus pseudomycoides; amino acidsequence.SEQ ID NO: 25: PC/PE-specific PLC, Bacillus pseudomycoides; amino acidsequence with N-terminal Alanine.SEQ ID NO: 26: PC/PE-specific PLC, Bacillus mycoides; coding sequence.SEQ ID NO: 27: PC/PE-specific PLC, Bacillus mycoides; amino acidsequence.SEQ ID NO: 28: PC/PE-specific PLC, Listeria innocua; coding sequence.SEQ ID NO: 29: PC/PE-specific PLC, Listeria innocua; amino acidsequence.SEQ ID NO: 30: PC/PE-specific PLC, Listeria innocua; amino acid sequencewith N-terminal Alanine.

SEQ ID NO: 31: SEQ ID NO: 3 of WO 2011/046812. SEQ ID NO: 32: SEQ ID NO:4 of WO 2011/046812.

SEQ ID NO: 33: Heterologous signal peptideSEQ ID NO: 34: PI-specific PLC, Amycolatopsis azurea; coding sequenceSEQ ID NO: 35: PI-specific PLC, Amycolatopsis azurea; amino acidsequenceSEQ ID NO: 36: PI-specific PLC, Amycolatopsis azurea; amino acidsequence with N-terminal Alanine.SEQ ID NO: 37: PC/PE-specific PLC, Bacillus macauensis; coding sequenceSEQ ID NO: 38: PC/PE-specific PLC, Bacillus macauensis; amino acidsequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to phospholipase C enzymes obtained fromvarious strains belonging to the genus pseudomonas which is anon-pathogenic (class 1) organism and therefore generally consideredsafe to work with in the laboratory. The phospholipase C enzymes derivedfrom pseudomonas strains all showed specificity towards PI.

The present invention also relates PC, PE-specific phospholipase Cenzymes which either are novel or which have never been expressed andcharacterized. The use of the PC, PE-specific phospholipase C,PurifinePLC, in degumming is known, it is however still relevant toidentify additional PC, PE-specific phospholipase C enzymes whichperform well in degumming. In a preferred embodiment the PC, PE-specificphospholipases are obtained from strains belonging to the genus ofBacillus or Listeria.

The present invention furthermore relates to a method for reducing thecontent of phospholipids in an oil composition using one or morebacterial phospholipase C enzymes. In particular when a PI-specificphospholipase C enzyme is combined with a PC, PE-specific phospholipaseC enzyme a beneficial effect is observed in relation to the removal ofphospholipids from an oil composition.

Polypeptides Having Phospholipase C Activity

An aspect of the present invention relates to a polypeptide havingphosphatidylinositol phospholipase C activity selected from the groupconsisting of: a) a polypeptide having at least 91% sequence identity tothe mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by apolynucleotide that hybridizes under medium stringency conditions withi) the mature polypeptide coding sequence of SEQ ID NO: 1, or ii) thefull-length complement of (i); c) a polypeptide encoded by apolynucleotide having at least 90% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1; d) a variant of the maturepolypeptide of SEQ ID NO: 2 comprising a substitution, deletion, and/orinsertion at one or more positions; and e) a fragment of the polypeptideof (a), (b), (c), or (d) that has phosphatidylinositol phospholipase Cactivity.

In one embodiment, the present invention relates to a polypeptide havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which hasspecificity towards phosphatidyl inositol. In one aspect, thepolypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2. In a preferredembodiment the mature polypeptide of SEQ ID NO: 2 corresponds to aminoacids 26 to 322 of SEQ ID NO: 2.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO: 2 or an allelic variant thereof; or is a fragmentthereof having phosphatidyl inositol phospholipase C activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the polypeptidecomprises or consists of amino acids 26 to 322 of SEQ ID NO: 2 or toamino acids 1 to 298 of SEQ ID NO: 3.

In particular, the polypeptide may have a length of 280-320 amino acidresidues, such as a length of 280-310 amino acid residues, 280-305 aminoacid residues, 280-300 amino acid residues, 280-298 amino acid residues,280-297 amino acid residues, 280-296 amino acid residues, 285-320 aminoacid residues, 285-315 amino acid residues, 285-310 amino acid residues,285-305 amino acid residues, 285-300 amino acid residues, 285-298 aminoacid residues, 285-297 amino acid residues, 285-296 amino acid residues,290-320 amino acid residues, 290-315 amino acid residues, 290-310 aminoacid residues, 290-305 amino acid residues, 290-300 amino acid residues,290-298 amino acid residues, 290-297 amino acid residues, 290-296 aminoacid residues, 295-320 amino acid residues, 295-315 amino acid residues,295-310 amino acid residues, 295-305 amino acid residues, 295-300 aminoacid residues, 295-298 amino acid residues, 255-297 amino acid residues,or a length of 295-296 amino acid residues.

In a preferred embodiment, the phosphatidylinositol phospholipase C ofthe invention is capable of reducing the PI content in a crude oil by atleast 30% when applied in 10 mg Enzyme Protein/kg oil at the optimal pHof the polypeptide, more preferred at least 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In a furtherembodiment the optimal pH range of the polypeptide of the presentinvention is between 4.5 to 8.5, more preferred from 5.0 to 8.0, evenmore preferred from 6.5 to 7.5.

According to some embodiments, the phosphatidylinositol phospholipase Cof the invention has a thermal denaturation temperature of at least 60°C., such as 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77°C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86°C., 87° C., 88° C., 89° C. or at least 90° C. as determined byDifferential Scanning calorimetry (DSC).

The denaturation temperature may in particular be determined as the topof denaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating a 1 mg/ml solution of the polypeptide in buffer(50 mM Na-acetate pH 5.5, or 50 mM Hepes pH 7) at a constant programmedheating rate of 200 K/hr.

According to some embodiments, the phosphatidylinositol phospholipase Cof the invention may be able to reduce the phosphatidylinositol contentof crude soy bean oil by 50% or more, 50%, such as by 55%, by 60%, by65%, by 70%, by 75%, by 80%, by 85%, by 90% or by 95% or more, thereduction in phosphatidylinositol content being determined by ³¹P-NMRafter addition of 100 mg enzyme protein (EP)/kg oil and incubation ofthe oil and enzyme at 50° C. for 2 hours at pH 5.5.

Preferably, the phosphatidylinositol phospholipase C of the invention isable to reduce the phosphorous content of crude soy bean oil to 20 mg/kgoil or less as determined by Inductively coupled plasma optical emissionspectrometry (ICP-OES) after incubation of 4 mg enzyme protein/kg oil ina low aqueous system comprising 3% water based on oil amount at 50-60°C. for 5 hours.

For the purpose of determining the ability of the polypeptide to reducephosphorous, the crude soy oil comprising 80-140 ppm phosphorous presentas phosphatidic acid (PA), 140-200 ppm phosphorous present asphosphatidyl ethanolamine (PE), 70-110 ppm phosphorous present asphosphatidic acid (PI) and 130-200 ppm phosphorous present asphosphatidyl choline may be used; the phosphorous content being measuredby ³¹P-NMR.

In further embodiments, the phosphatidylinositol phospholipase C of theinvention is robust towards varying pH conditions and shows goodperformance in water degumming (no acid/base) as well as acid assisteddegumming followed by base neutralization with varying concentrations ofNaOH (e.g., Acid/base pretreatment by addition of Ortho Phosphoric acidin amounts equal to 0.05% (100% pure Ortho Phosphoric acid) based on oilamount followed by base neutralization with 0.5 eqv, 1.0 eqv or 1.5 eqvNaOH).

Further, the reduction of phosphorous content may be obtained in an oildegumming process comprising the steps of:

i) Optionally treating crude soy bean oil with acid/base by adding an85% solution of Ortho Phosphoric acid in amounts corresponding to 0.05%(100% pure Ortho Phosphoric acid) based on oil amount, mixing inultrasonic bath for 5 minutes, followed by incubation in rotator for 15minutes and base neutralization with 4 M NaOH applied in equivalents(from 0.5 to 0.15) to pure Ortho Phosphoric acid in ultrasonic bath for5 minutes;

ii) Adding the polypeptide to the oil in amounts of 4 mg enzymeprotein/kg oil in a low aqueous system comprising 3% water based on oilamount and subjecting the oil and the polypeptide to ultrasonictreatment for 5 minutes;

iii) Incubating the polypeptide and oil at 50-60° C. for 5 hours withstirring at 20 rpm;

iv) Centrifuging the oil and the polypeptide at 700 g at 85° C. for 15minutes.

Homologues of PI-specific phospholipase C of the present invention havebeen identified from sequences annotated in genome sequencing projects.When aligned to the mature sequence of SEQ ID NO: 2 the identities areas follows:

SEQ ID NO: 2 mature 100.00 J2E6B1 (SEQ ID NO: 5) 83.11 J3EBR2 (SEQ IDNO: 8) 82.78 I4Y4N5 (SEQ ID NO: 11) 82.45 Q4K3U9 (SEQ ID NO: 14) 90.07R4RTF9 (SEQ ID NO: 17) 90.73 M2NSV3 (SEQ ID NO: 35) 20.00

To our knowledge none of these homologues have ever been expressed andcharacterized and their use in degumming or any other application hasnever been described. For the purpose of generating nucleic acidconstructs, expression vectors, and host cells as well as compositionsand methods of use the PI-specific phospholipase C of SEQ ID NO: 2 aswell as the homologues of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQID NO: 14 and SEQ ID NO: 17 are PI-specific phospholipase C polypeptidesof the present invention and the polynucleotides encoding them arepolynucleotides of the present invention.

In another embodiment, the present invention relates to a polypeptidehaving PI-specific phospholipase C activity encoded by a polynucleotidethat hybridizes under medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1 or (ii) the full-length complement of (i) or (ii) (Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.). In an embodiment, the polypeptide has beenisolated.

The polynucleotides of SEQ ID NO: 1, 4, 7, 10, 13 or 16 or a subsequencethereof, as well as the polypeptide of SEQ ID NO: 2, 5, 8, 11, 14 or 17or a fragment thereof may be used to design nucleic acid probes toidentify and clone DNA encoding polypeptides having PI-specificphospholipase C activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic DNA or cDNA of a cell ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having PI-specific phospholipase C activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or asubsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under medium to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In another embodiment, the present invention relates to a polypeptidehaving PI-specific phospholipase C activity encoded by a polynucleotidehaving a sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1 at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In a further embodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. A specificvariant of SEQ ID NO: 2 is disclosed as SEQ ID NO: 3 containing an Ainserted in front of the Q in position 26 of SEQ ID NO: 2. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changesmay be of a minor nature, that is conservative amino acid substitutionsor insertions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of 1-30 amino acids;small amino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Another aspect of the present invention relates to a polypeptide havingPC and PE-specific phospholipase C activity, selected from the groupconsisting of: a) a polypeptide having at least 70% sequence identity tothe mature polypeptide of SEQ ID NO: 19; b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with i)the mature polypeptide coding sequence of SEQ ID NO: 18, or ii) thefull-length complement of (i); c) a polypeptide encoded by apolynucleotide having at least 70% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 18; d) a variant of the maturepolypeptide of SEQ ID NO: 19 comprising a substitution, deletion, and/orinsertion at one or more positions; and e) a fragment of the polypeptideof (a), (b), (c), or (d) that has PC and PE specific phospholipase Cactivity.

In one embodiment, the present invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 19 of atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have PC and PE-specific phospholipase Cactivity. In one aspect, the polypeptides differ by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide of SEQ ID NO: 19.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO: 19 or an allelic variant thereof; or is afragment thereof having phospholipase C activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 19. In another aspect, the polypeptide comprises or consists ofamino acids 34 to 278 of SEQ ID NO: 19 or amino acid residues 1-246 ofSEQ ID NO: 20.

In further embodiments, the polypeptide has a length of 220-280 aminoacid residues, such as a length of 220-270 amino acid residues, 220-260amino acid residues, 220-250 amino acid residues, 220-248 amino acidresidues, 220-246 amino acid residues, 220-244 amino acid residues,225-280 amino acid residues, 225-270 amino acid residues, 225-260 aminoacid residues, 225-250 amino acid residues, 225-248 amino acid residues,225-246 amino acid residues, 225-244 amino acid residues, 230-280 aminoacid residues, 230-270 amino acid residues, 230-260 amino acid residues,230-250 amino acid residues, 230-248 amino acid residues, 230-246 aminoacid residues, 230-244 amino acid residues, 235-280 amino acid residues,235-270 amino acid residues, 235-260 amino acid residues, 235-250 aminoacid residues, 235-248 amino acid residues, 235-246 amino acid residues,235-244 amino acid residues, 240-280 amino acid residues, 240-270 aminoacid residues, 240-260 amino acid residues, 240-250 amino acid residues,240-248 amino acid residues, 240-246 amino acid residues, 240-244 aminoacid residues, 242-280 amino acid residues, 242-270 amino acid residues,242-260 amino acid residues, 242-250 amino acid residues, 242-248 aminoacid residues, 242-246 amino acid residues, 242-244 amino acid residues,243-280 amino acid residues, 243-270 amino acid residues, 243-260 aminoacid residues, 243-250 amino acid residues, 243-248 amino acid residues,243-246 amino acid residues, 243-244 amino acid residues.

The polypeptide having PC and PE-specific phospholipase C activity mayin particular have a thermal denaturation temperature of at least 60°C., such as 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77°C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86°C., 87° C., 88° C., 89° C. or at least 90° C. as determined byDifferential Scanning calorimetry (DSC).

The said denaturation temperature may be determined as the top ofdenaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating a 1 mg/ml solution of the polypeptide in buffer(50 mM Na-acetate pH 5.5, or 50 mM Hepes pH 7) at a constant programmedheating rate of 200 K/hr.

In a preferred embodiment, the PC and PE-specific phospholipase C of theinvention is capable of reducing the PC and PE content in a crude oil.Preferably, the polypeptide of the invention is capable of reducing thePC and PE content in a crude oil by at least 30% each when applied in 10mg Enzyme Protein/kg oil at the optimal pH of the polypeptide, morepreferred at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or 100%. In a further embodiment the optimal pH rangeof polypeptide of the present invention is between 5.0 to 8.5, morepreferred from 5.5 to 8.0, even more preferred from 6.0 to 7.5. Infurther embodiments the polypeptide having PC and PE-specificphospholipase C activity is able to reduce the phosphatidyl ethanolamineand/or phosphatidyl choline content of crude soy bean oil by 50% ormore, 50%, such as by 55%, by 60%, by 65%, by 70%, by 75%, by 80%, by85%, by 90% or by 95% or more, the reduction in phosphatidylethanolamine and/or phosphatidyl choline content being determined by³¹P-NMR after addition of 100 mg enzyme protein (EP)/kg oil andincubation of the oil and enzyme at 50° C. for 2 hours at pH 5.5.

In still further embodiments, the polypeptide having PC and PE-specificphospholipase C activity is able to reduce the phosphorous content ofcrude soy bean oil to 20 mg/kg oil or less as determined by Inductivelycoupled plasma optical emission spectrometry (ICP-OES) after incubationof 4 mg enzyme protein/kg oil in a low aqueous system comprising 3%water based on oil amount at 50-60° C. for 5 hours.

The ability of the polypeptide to reduce phosphorous content may inparticular be determined using a crude soy oil which comprises 80-140ppm phosphorous present as phosphatidic acid (PA), 140-200 ppmphosphorous present as phosphatidyl ethanolamine (PE), 70-110 ppmphosphorous present as phosphatidic acid (PI) and 130-200 ppmphosphorous present as phosphatidyl choline; the phosphorous contentbeing measured by ³¹P-NMR.

In particular, the reduction of phosphorous content and/or reduction inphosphatidyl ethanolamine and/or phosphatidyl choline content may beobtained in an oil degumming process comprising the steps of:

i) Optionally treating crude soy bean oil with acid/base by adding an85% solution of Ortho Phosphoric acid in amounts corresponding to 0.05%(100% pure Ortho Phosphoric acid) based on oil amount, mixing inultrasonic bath for 5 minutes, followed by incubation in rotator for 15minutes and base neutralization with 4 M NaOH applied in equivalents(from 0.5 to 0.15) to pure Ortho Phosphoric acid in ultrasonic bath for5 minutes;

ii) Adding the polypeptide to the oil in amounts of 4 mg enzymeprotein/kg oil in a low aqueous system comprising 3% water based on oilamount and subjecting the oil and the polypeptide to ultrasonictreatment for 5 minutes;

iii) Incubating the polypeptide and oil at 50-60° C. for 5 hours withstirring at 20 rpm;

iv) Centrifuging the oil and the polypeptide at 700 g at 85° C. for 15minutes.

The closest related sequence to the PC, PE-specific PLC of the presentinvention is SEQ ID NO: 4 from WO2011/046812 (SEQ ID NO: 32 in thisapplication) with 44.6% identity to the mature sequence of SEQ ID NO: 19(amino acids 34 to 278). This as well as the following PC, PE-specificPLC's identified by UniProt number may be useful in the method of thepresent invention

SEQ ID NO: 19 mature 100.00 44.63 43.57 43.80 43.80 31.65 SEQ ID NO: 3244.63 100.00 80.14 80.50 80.50 40.23 C3HDV6 (SEQ ID NO: 22) 43.57 80.14100.00 88.69 88.34 40.44 P34B3F (SEQ ID NO: 24) 43.80 80.50 88.69 100.0098.59 39.05 C3AHL7 (SEQ ID NO: 27) 43.80 80.50 88.34 98.59 100.00 39.05E3Z3X0 (SEQ ID NO: 29) 31.65 40.23 40.44 39.05 39.05 100.00 I8AFV4 (SEQID NO: 38) 42.08

The mature sequence of SEQ ID NO: 22, corresponding to amino acids 39 to283 has also been disclosed in relation to degumming as SEQ ID NO: 5 inEP 1788080. To our knowledge none of the PC, PE-specific PLC's of SEQ IDNO: 24, 27 or 29 have ever been expressed and characterized and theiruse in degumming or any other application has never been described. Forthe purpose of generating nucleic acid constructs, expression vectors,and host cells as well as compositions and methods of use the PC,PE-specific phospholipase C of SEQ ID NO: 19 as well as the homologuesof SEQ ID NO: 24, SEQ ID NO: 27 and SEQ ID NO: 29 are PC, PE-specificphospholipase C polypeptides of the present invention and thepolynucleotides encoding them are polynucleotides of the presentinvention.

In another embodiment, the present invention relates to a polypeptidehaving PC and PE-specific phospholipase C activity encoded by apolynucleotide that hybridizes under low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 18 or (ii) thefull-length complement of (i) or (ii) (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Inan embodiment, the polypeptide has been isolated.

The polynucleotide of SEQ ID NO: 18 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 19 or a fragment thereof may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having phospholipase C activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicDNA or cDNA of a cell of interest, following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having PC and PE-specific phospholipase Cactivity. Genomic or other DNA from such other strains may be separatedby agarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 18 or a subsequence thereof, the carrier material isused in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 18; (ii) the mature polypeptide coding sequence of SEQID NO: 18; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In another embodiment, the present invention relates to a polypeptidehaving PC and PE-specific phospholipase C activity encoded by apolynucleotide having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 18 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%. In afurther embodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 19 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. A specificvariant of SEQ ID NO: 19 is disclosed as SEQ ID NO: 20 containing an Ainserted in front of the W in position 34 of SEQ ID NO: 19. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 19 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changesmay be of a minor nature, that is conservative amino acid substitutionsor insertions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of 1-30 amino acids;small amino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

In relation to the polypeptides of the present invention examples ofconservative substitutions are within the groups of basic amino acids(arginine, lysine and histidine), acidic amino acids (glutamic acid andaspartic acid), polar amino acids (glutamine and asparagine),hydrophobic amino acids (leucine, isoleucine and valine), aromatic aminoacids (phenylalanine, tryptophan and tyrosine), and small amino acids(glycine, alanine, serine, threonine and methionine). Amino acidsubstitutions that do not generally alter specific activity are known inthe art and are described, for example, by H. Neurath and R. L. Hill,1979, In, The Proteins, Academic Press, New York. Common substitutionsare Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val,Ala/Glu, and Asp/Gly.

Other possible approaches to generating variants having similar orsubstantially similar physic-chemical or functional properties as thePI-specific phospholipase C of SEQ ID NO: 2 (the mature polypeptide ofSEQ ID NO: 2) or the PC PE-specific phospholipase C of SEQ ID NO: 19(the mature polypeptide of SEQ ID NO: 19) would include introducingchanges in the amino acid sequence within regions showing medium to highvariability, identified by aligning the respective polypeptide withrelated sequences. In the PI-specific phospholipase C of SEQ ID NO: 2,such regions may be identified by alignment to best possible fit withthe amino acid sequences of SEQ ID NOs: 8, (UniProtKB/TrEMBL: J3EBR2),11 (UniProtKB/TrEMBL: 14Y4N5), 14 (UniProtKB/TrEMBL: Q4K3U9) and 17(UniProtKB/TrEMBL: R4RTF9).

By such alignment the following regions having medium or highvariability may be identified in SEQ ID NO: 2 (using the amino acidnumbering of SEQ ID NO: 2):

Amino acids 28-43 High variability Amino acid 59 High variability Aminoacids 82-88 High variability Amino acids 130-131 High variability Aminoacid 165 High variability Amino acids 254-255 High variability Aminoacid 266 High variability Amino acids 298-301 High variability Aminoacids 311-314 High variability

Hence, in some embodiments of the invention a polypeptide having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which hasspecificity towards phosphatidyl inositol, wherein one or more aminoacids residues have been substituted, deleted or added in one or more ofthe regions defined by amino acids 28-43, amino acid 59, amino acids82-88, amino acids 130-131, amino acid 266, amino acids 298-301, aminoacids 311314, using the amino acids numbering in SEQ ID NO: 2.

In further embodiments, these polypeptides differ by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide of SEQ ID NO: 2.

In corresponding embodiments relating to the PE/PC-specificphospholipase C of SEQ ID NO: 19, such regions may be identified byalignment to best possible fit with the amino acid sequences of SEQ IDNOs: 22, (UniProtKB/TrEMBL:C3HDV6), 24 (UniProtKB/TrEMBL:C3BG10), 27(UniProtKB/TrEMBL:C3AHL7) and 29 (UniProtKB/TrEMBL: E3Z3×0).

By such alignment the following regions having medium or highvariability may be identified in SEQ ID NO: 19 (using the amino acidnumbering of SEQ ID NO: 19):

Amino acids 3-72 High variability Amino acids 74-123 Medium variabilityAmino acids 129-142 High variability Amino acids 145-147 Mediumvariability Amino acids 154-155 Medium variability Amino acid 164-189High variability Amino acids 188-201 High variability Amino acids203-226 Medium variability Amino acids 228-237 High variability Aminoacids 244-246 Medium variability Amino acids 248-258 Medium variabilityAmino acids 260-278 Medium variability

In one embodiment, the present invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 19 of atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have PC and PE-specific phospholipase Cactivity, wherein one or more amino acids residues, such as up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, have beensubstituted, deleted or added in one or more of the regions defined byamino acids 3-72, amino acids 74-123, amino acids 129-142, amino acids145-147, amino acids 154-155, amino acids 164-189, amino acids 188-201,amino acids 203-226, amino acids 228-237, amino acids 244-246, aminoacids 248-258, amino acids 260-278, using the amino acid numbering inSEQ ID NO: 19.

In further embodiments, these polypeptides differ by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide of SEQ ID NO: 19.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for the desired phospholipase C activity toidentify amino acid residues that are critical to the activity of themolecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708.The active site of the enzyme or other biological interaction can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; WIodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Essential amino acids in the sequence of amino acids 26 to 322 of SEQ IDNO: 2 are predicted to be located at positions H50, N51, and D74. Theseamino acids are believed to be involved in coordinating the calcium inthe active site. The prediction is supported by the following articleIwasaki et al., 1998, Biochimica et Biophysica Acta 1391: 52-66 thatshow that when the H corresponding to H50 in SEQ ID NO: 2 is changed thePLC identified as UniProt B3A043 or PDB 3H4X dies. In a preferredembodiment a polypeptide of the invention maintains the amino acidscorresponding to position 50, 51 and 74 when aligned to SEQ ID NO: 2.

Essential amino acids in the sequence of amino acids 34 to 278 of SEQ IDNO: 19 are predicted to be located at positions W34, H47, D88, H102,H152, D156, H162, H177 and E181. These amino acids are believed to beinvolved in coordinating the three Zn ions need for the catalyticactivity based on a homology model of the sequence. In a preferredembodiment a polypeptide of the invention maintains the amino acidscorresponding to positions 34, 47, 88, 102, 152, 156, 162, 177 and 181when aligned to SEQ ID NO: 19.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Phospholipase C Activity

A polypeptide having phospholipase C activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, thepolypeptide may be a Gram-positive bacterial polypeptide such as aBacillus, Listeria or Pseudomonas polypeptide.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspseudomycoides, Bacillus pumilus, Bacillus stearothermophilus, Bacillussubtilis, or Bacillus thuringiensis polypeptide.

In another aspect, the polypeptide is a Listeria innocua polypeptide.

In another aspect, the polypeptide is a Pseudomonas chlororaphis orPseudomonas protegens polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to polynucleotides encoding apolypeptide of the present invention. In an embodiment, thepolynucleotide encoding the polypeptide of the present invention hasbeen isolated.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Bacillus or Pseudomonas,or a related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of thepolynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:18, e.g., a subsequence thereof, and/or by introduction of nucleotidesubstitutions that do not result in a change in the amino acid sequenceof the polypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence inan expression host. Preferably the expression is done in a suitable hostcell under conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart. In a preferred embodiment the polynucleotide encodes an alanine infront of the sequence encoding a mature a phospholipase C. In a furtherembodiment the mature phospholipase C contain an N-terminal sequencestarting with the amino acids WSA, and the polypeptide expressed fromthe polynucleotide will result in an N-terminal sequence starting withamino acids AWSA.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. Other examples of regulatory sequences are those that allow forgene amplification.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. Preferably the polynucleotide is heterologous,meaning that it does not exist naturally in the host cell. A constructor vector comprising a polynucleotide is introduced into a host cell sothat the construct or vector is maintained as a chromosomal integrant oras a self-replicating extra-chromosomal vector as described earlier. Theterm “host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.In a preferred embodiment the host cell is a recombinant host cell whichdoes not exist in nature.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

Methods of Production

Based on the nucleotide sequence identified as SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ IDNO: 31 a synthetic gene can be obtained from a number of vendors such asGene Art (GENEART AG BioPark, Josef-Engert-Str. 11, 93053, Regensburg,Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite E, Menlo Park,Calif. 94025, USA). The synthetic gene can be designed to incorporateadditional DNA sequences such as restriction sites or homologousrecombination regions to facilitate cloning into an expression vector asdescribed above and expressed in a host cell as described above, forexample in Bacillus subtilis.

An aspect of the present invention relates to a method of producing aphospholipase C polypeptide in a bacterial host, in particular, aBacillus host, more specifically a Bacillus subtilis or Bacilluslicheniformis host, wherein the phospholipase C coding sequence encodean alanine in front of the predicted N-terminal amino acid of thephospholipase C polypeptide. Preferably the phospholipase C is a PC andPE-specific phospholipase C polypeptide. This will result in a maturephospholipase C polypeptide with an N-terminal alanine as exemplified inSEQ ID NO: 20 and SEQ ID NO: 30. Preferably the wild type maturesequence of the phospholipase C has an N-terminal sequences startingwith WSA. Without being bound by theory the inventors believe that thisextra alanine protect the N-terminal sequence of the maturephospholipase C sequence, e.g., amino acids WSA, from protease activityin the host cell.

The present invention also relates to methods of producing aphosphatidylinositol phospholipase C polypeptide of the presentinvention, comprising (a) cultivating a cell, which in its wild-typeform produces the polypeptide, under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide. Inone aspect, the cell is a Pseudomonas cell.

The present invention also relates to methods of producing a PC andPE-specific phospholipase C polypeptide of the present invention,comprising (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and optionally, (b) recovering the polypeptide. In oneaspect, the cell is a Bacillus cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide with phospholipase C activity may be detected usingmethods known in the art, see the “Assay for phospholipase activity”section below. These detection methods include, but are not limited to,use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate, e.g., P-NMR assay described inexample 5 or liquid chromatography coupled to triple quadrupole massspectrometer (LC/MS/MS) as described in Example 7 or lecithin plateassays.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Assays for Phospholipase Activity

The invention provides isolated, synthetic or recombinant polypeptides(e.g., enzymes, antibodies) having a phospholipase activity, or anycombination of phospholipase activities, and nucleic acids encodingthem. Any of the many phospholipase activity assays known in the art canbe used to determine if a polypeptide has a phospholipase activity andis within the scope of the invention. Routine protocols for determiningphospholipase A, B, D and C, are well known in the art.

Exemplary activity assays include turbidity assays, methylumbelliferylphosphocholine (fluorescent) assays, Amplex red (fluorescent)phospholipase assays, thin layer chromatography assays (TLC), cytolyticassays and p-nitrophenylphosphorylcholine assays.

Using these assays polypeptides, peptides or antibodies can be quicklyscreened for a phospholipase activity.

Plate assays with a substrate containing agar can be used to determinephospholipase activity Plate assay. The assay can be conducted asfollows. Plates are casted by mixing of 5 ml 2% Agarose (Litex HSA 1000)prepared by mixing and cooking in buffers (100 mM HEPES and 100 mMCitrate with pH adjusted from pH 3.0 to pH 7.0) for 5 minutes followedby cooling to approximately 60° C. and 5 ml substrate(L-α-phosphatidylcholine, 95% from Soy (Avanti 441601) orL-α-phosphatidylinositol from Soy (Avanti 840044P) for PI-specificity orL-α-phosphatidylethanolamine from Soy (Avanti 840024P) dispersed inwater (MilliQ) at 60° C. for 1 minute with Ultra Turrax forPC-specificity) gently mixed into petri dishes with diameter of 7 cm andcooled to room temperature before holes with a diameter of approximately3 mm were punched by vacuum. Ten microliters of purified enzyme dilutedto 0.4 mg/ml is added into each well before plates were sealed byparafilm and placed in an incubator at 55° C. for 48 hours. Plates weretaken out for photography at regular intervals.

Turbidity assays to determine phospholipase activity are described,e.g., in Kauffmann, 2001, “Conversion of Bacillus thermocatenulatuslipase into an efficient phospholipase with increased activity towardslong-chain fatty acyl substrates by directed evolution and rationaldesign,” Protein Engineering 14:919-928; Ibrahim, 1995, “Evidenceimplicating phospholipase as a virulence factor of Candida albicans,”Infect. Immun. 63:1993-1998.

Methylumbelliferyl (fluorescent) phosphocholine assays to determinephospholipase activity are described, e.g., in Goode, 1997, “Evidencefor cell surface internal phospholipase activity in ascidian eggs,”Develop. Growth Differ. 39:655-660; Diaz, 1999, “Directfluorescence-based lipase activity assay,” Bio Techniques 27:696-700.

Amplex Red (fluorescent) Phospholipase Assays to determine phospholipaseactivity are available as kits, e.g., the detection ofphosphatidylcholine-specific phospholipase using an Amplex Redphosphatidylcholine-specific phospholipase assay kit from MolecularProbes Inc. (Eugene, Oreg.), according to manufacturer's instructions.

Fluorescence is measured in a fluorescence microplate reader usingexcitation at 560±10 nm and fluorescence detection at 590±10 nm. Theassay is sensitive at very low enzyme concentrations.

Thin layer chromatography assays (TLC) to determine phospholipaseactivity are described, e.g., in Reynolds, 1991, Methods in Enzymol.197:3-13; Taguchi, 1975, “Phospholipase from Clostridium novyi typeA.I,” Biochim. Biophys. Acta 409:75-85. Thin layer chromatography (TLC)is a widely used technique for detection of phospholipase activity.Various modifications of this method have been used to extract thephospholipids from the aqueous assay mixtures. In some PLC assays thehydrolysis is stopped by addition of chloroform/methanol (2:1) to thereaction mixture. The unreacted starting material and the diacylglycerolare extracted into the organic phase and may be fractionated by TLC,while the head group product remains in the aqueous phase. For moreprecise measurement of the phospholipid digestion, radio labeledsubstrates can be used (see, e.g., Reynolds, 1991, Methods in Enzymol.197:3-13). The ratios of products and reactants can be used to calculatethe actual number of moles of substrate hydrolyzed per unit time. If allthe components are extracted equally, any losses in the extraction willaffect all components equally. Separation of phospholipid digestionproducts can be achieved by silica gel TLC withchloroform/methanol/water (65:25:4) used as a solvent system (see, e.g.,Taguchi, 1975, Biochim. Biophys. Acta 409:75-85).

p-N itrophenylphosphorylcholine assays to determine phospholipaseactivity are described, e.g., in Korbsrisate, 1999, J. Clin. Microbiol.37:3742-3745; Berka, 1981, Infect. Immun. 34:1071-1074. This assay isbased on enzymatic hydrolysis of the substrate analogp-nitrophenylphosphorylcholine to liberate a yellow chromogenic compoundp-nitrophenol, detectable at 405 nm. This substrate is convenient forhigh throughput screening. Similar assays using substrates towards theother phospholipid groups can also be applied, e.g., usingp-nitrophenylphosphorylinositol or p-nitrophenylphosphorylethanolamine.

A cytolytic assay can detect phospholipases with cytolytic activitybased on lysis of erythrocytes. Toxic phospholipases can interact witheukaryotic cell membranes and hydrolyze phosphatidylcholine andsphingomyelin, leading to cell lysis. See, e.g., Titball, 1993,Microbiol. Rev. 57:347-366.

Further assays like ³¹P-NMR and Liquid Chromatography coupled to triplequadrupole mass spectrometer (LC/MS/MS) are described in the examplesection of this application.

Compositions

The present invention also relates to compositions comprising aPI-specific PLC polypeptide of the present invention, preferably with anadditional component. The PI-specific PLC polypeptides of the presentinvention includes a) a polypeptide comprising an amino acid sequenceselected from the group consisting of: i) amino acid residues 26-322 ofSEQ ID NO: 2 or amino acid residues 1-298 of SEQ ID NO: 3; ii) aminoacid residues 26-323 of SEQ ID NO: 5 or amino acid residues 1-299 of SEQID NO: 6; iii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acidresidues 1-299 of SEQ ID NO: 9; iv) amino acid residues 26-323 of SEQ IDNO: 11 or amino acid residues 1-296 of SEQ ID NO: 12; v) amino acidresidues 26-322 of SEQ ID NO: 14 or amino acid residues 1-298 of SEQ IDNO: 15; vi) amino acid residues 26-322 of SEQ ID NO: 17; and vii) aminoacid residues 28-339 of SEQ ID NO: 35; or b) a polypeptide comprising anamino acid sequence which has at least 75% identity to one of the aminoacid sequences in a); or c) a functional fragment of a) or b).

The present invention also relates to compositions comprising a PC,PE-specific PLC polypeptide of the present invention, preferably with anadditional component.

The present invention also relates to compositions comprising a mixtureof a PI-specific PLC of the present invention with one or more, furtherphospholipase activities selected from PLA1, PLA2, PLC and PLD.

The present invention also relates to a composition comprising a mixtureof a phosphatidylinositol phospholipase C from the genus of Pseudomonasand a PC and PE-specific phospholipase C polypeptide. A preferredcomposition of the invention comprises a PI-specific PLC polypeptide ofthe present invention combined with a PC, PE-specific PLC polypeptide.Preferably the PC and PE-specific PLC is selected from Purifine or amature polypeptide of SEQ ID NO: 19, 20, 22, 24, 25, 27, 29, 30 32 or38. The PC and PE-specific PLC may also be selected from a sequencewhich is at least 80%, 85%, 90%, 95% or 98% identical to a maturepolypeptide of SEQ ID NO: 19, 20, 22, 24, 25, 27, 29, 30 32 or 38 whichhas PC and PE-specificity. In alternative embodiments of the inventionthe composition comprises a PI-specific PLC polypeptide of the presentinvention combined with a PLC with specificity towards PA or PC or PE,or PE and PA; or PC and PA; or PC and PE and PA; or any combinationthereof.

The phospholipases of the present invention may be formulated withcomponents selected from the group consisting of buffer agents,inorganic salts, solvents, inert solids and mixtures thereof.Appropriate buffer systems, e.g., are made from aqueous solutions ofsalts or organic acids, amino acids, phosphate, amines or ammonia inconcentrations between 0.01 M and 1 M at pH 2 to 10. Preferably, alkalimetal salts of citric acid, acetic acid, glycine and/or hydrochloridesof tris(hydroxymethyl)amine and ammonia at 0.1 M to 0.2 M at pH 4 to 8are used. Preferably, the phospholipase is dissolved in an aqueousbuffer solution such as glycine buffer, citric acid buffer, etc. Citratecontaining buffers have been found to be very suitable, in particular,sodium citrate buffers, preferably at neutral pH.

The compositions of the invention may comprise phospholipases of theinvention immobilized unto a solid support. Solid supports useful inthis invention include gels. Some examples of gels include Sepharose,gelatin, glutaraldehyde, chitosan-treated glutaraldehyde,albumin-glutaraldehyde, chitosan-Xanthan, toyopearl gel (polymer gel),alginate, alginate-polylysine, carrageenan, agarose, glyoxyl agarose,magnetic agarose, dextranagarose, poly(Carbamoyl Sulfonate) hydrogel,BSA-PEG hydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.Another type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, Si02,Ah03. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porous glass, aminopropylglass or any combination thereof. Another type of solid support that canbe used is a microelectrode. An example is a polyethyleneimine-coatedmagnetite. Graphitic particles can be used as a solid support. Otherexemplary solid supports used to practice the invention comprisediatomaceous earth products and silicates. Some examples include CELITE®KENITE®, DIACTIV®, PRIM ISIL® ′ DIAFIL® diatomites and MICRO-CEL®′CALFLO®, SILASORB™, and CELKA TE® synthetic calcium and magnesiumsilicates.

Some examples of methods for immobilizing enzymes include, e.g.,electrostatic droplet generation, electrochemical means, via adsorption,via covalent binding, via cross-linking, via a chemical reaction orprocess, via encapsulation, via entrapment, via calcium alginate, or viapoly (2-hydroxyethyl methacrylate). Like methods are described inMethods in Enzymology, Immobilized Enzymes and Cells, Part C. 1987.Academic Press. Edited by S. P. Colowick and N. 0. Kaplan. Volume 136;and Immobilization of Enzymes and Cells. 1997. Humana Press. Edited byG. F. Bickerstaff. Series: Methods in Biotechnology, Edited by J. M.Walker.

In a further embodiment the composition may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of hydrolase, isomerase, ligase, lyase,oxidoreductase, or transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The phospholipases or compositions of the invention may be applied in aprocess for removing phospholipids from an oil, e.g., a vegetable oil,animal oil or fat, tallow, or grease.

Applications in which the phospholipase of the invention can be usedcomprise i) degumming of oil, e.g., vegetable oil, or an ediblevegetable oil, or in a process comprising hydrolysis of phospholipids inthe gum fraction from water degumming to release entrapped triglycerideoil, ii) in a process comprising hydrolysis of phospholipids to obtainimproved phospholipid emulsifiers, in particular wherein saidphospholipid is lecithin, iii) in a process for improving thefilterability of an aqueous solution or slurry of carbohydrate originwhich contains phospholipid, iv) in a process for the extraction of oil,v) in a process for the production of an animal feed product, vi) in aprocess for the production of a biofuel, e.g., a biodiesel, vii) in aprocess for the production of a detergent product, and/or viii) in aprocess for making a baked product, comprising adding the phospholipaseto a dough, and baking the dough to make the baked product.

The phospholipases of the invention may be applied in a processcomprising treatment of a phospholipid or lysophospholipid with thephospholipases or compositions of the invention.

The phospholipases or compositions react with the phospholipids orlysophospholipid to form monoglyceride or diglyceride and a phosphateester or phosphoric acid.

Degumming: The phospholipases of the invention and combinations thereofmay be used for degumming oil, e.g., animal oil or fat, tallow, greaseor a vegetable oil, i.e., in a process to reduce the phospholipidcontent in the oil. The degumming process is applicable to thepurification of any edible oil which contains phospholipid, e.g.,vegetable oil such as soybean oil, rape seed oil, or sunflower oil orany other oil mentioned under the definition of crude oils.

PI-specific PLC converts phosphatidyl inositol (PI) to diglyceride andphosphoinositol. PC-specific PLC converts phosphatidylcholine (PC) todiglyceride and phosphocholine. PE-specific PLC convertsphosphatidylethanolamine (PE) to diglyceride and phophoethanolamine. Thediglyceride stays in the oil phase (improving oil yield) and thephosphorous-containing moieties separates into the aqueous phase whereit is removed as a component of the heavy phase during centrifugation.The gum phase (heavy phase) may be treated further with a phospholipaseor composition of the present invention to increase hydrolysis ofphospholipids in the gum fraction from water degumming to releaseentrapped triglyceride oil This is particular useful when de degummingprocess has not already applied phospholipases. Phospholipases of theinvention, e.g., a PI-specific PLC's and/or PC, PE-specific PLC's of theinvention, can be incorporated into either water degumming or a chemicalor physical oil refining process. In a preferred embodiment thephospholipases of the invention are incorporated into a water degummingprocess with preferably less than 10%, 9%, 8%, 7%, 6% or 5% water, evenmore preferably less than 4%, 3% or 2% water, preferably at 50° C. orabove, even more preferably at 60° C. or above.

In another preferred embodiment, the phospholipases of the invention areincorporated into a physical refining process applying citric acid orphosphoric acid and sodium hydroxide to facilitate hydratability ofinsoluble phospholipids and ensure an environment suitable for theenzyme with preferably less than 0.15% citric acid or phosphoric acid,even more preferably less than 0.1%, 0.09%, 0.08%, 0.07%, 0.06% or0.05%; and less than 4%, 3% or 2% water, preferably at 50° C. or above,even more preferably at 60° C. or above.

In other embodiments, the degumming process is a caustic refiningprocess or acid degumming process.

An aspect of the present invention is a method for reducing the contentof phospholipids in an oil composition, the method comprising a)providing an oil composition containing a quantity of phospholipids, b)contacting said oil composition with a phosphatidylinositolphospholipase C (PI-specific PLC), and a PC and PE-specificphospholipase C under conditions sufficient for the enzymes to reactwith the phospholipids to create diglyceride and phosphate ester, and,c) separating the phosphate ester from the oil composition. In apreferred embodiment the phosphatidylinositol phospholipase C is fromthe genus of Pseudomonas. In other preferred embodiments, the oil issubject to acid/base treatment prior to being contacted with thephospholipase(s).

In a preferred embodiment, the phosphatidylinositol phospholipase C isa) a polypeptide comprising an amino acid sequence selected from thegroup consisting of: i) amino acid residues 26-322 of SEQ ID NO: 2 oramino acid residues 1-298 of SEQ ID NO: 3; ii) amino acid residues26-323 of SEQ ID NO: 5 or amino acid residues 1-299 of SEQ ID NO: 6;iii)amino acid residues 26-323 of SEQ ID NO: 8 or amino acid residues1-299 of SEQ ID NO: 9; iv) amino acid residues 26-323 of SEQ ID NO: 11or amino acid residues 1-296 of SEQ ID NO: 12; v) amino acid residues26-322 of SEQ ID NO: 14 or amino acid residues 1-298 of SEQ ID NO: 15;vi)amino acid residues 26-322 of SEQ ID NO: 17; and vii) amino acidresidues 28-339 of SEQ ID NO: 35; or b) a polypeptide comprising anamino acid sequence which has at least 75% identity to one of the aminoacid sequences in a); or c) a functional fragment of a) or b).

In some embodiments, said phosphatidylinositol phospholipase C is apolypeptide having a length of 280-320 amino acid residues, such as alength of 280-310 amino acid residues, 280-305 amino acid residues,280-300 amino acid residues, 280-298 amino acid residues, 280-297 aminoacid residues, 280-296 amino acid residues, 285-320 amino acid residues,285-315 amino acid residues, 285-310 amino acid residues, 285-305 aminoacid residues, 285-300 amino acid residues, 285-298 amino acid residues,285-297 amino acid residues, 285-296 amino acid residues, 290-320 aminoacid residues, 290-315 amino acid residues, 290-310 amino acid residues,290-305 amino acid residues, 290-300 amino acid residues, 290-298 aminoacid residues, 290-297 amino acid residues, 290-296 amino acid residues,295-320 amino acid residues, 295-315 amino acid residues, 295-310 aminoacid residues, 295-305 amino acid residues, 295-300 amino acid residues,295-298 amino acid residues, 255-297 amino acid residues, or a length of295-296 amino acid residues.

According to other embodiments, the said PC and PE-specificphospholipase C is a polypeptide having a length of 220-280 amino acidresidues, such as a length of 220-270 amino acid residues, 220-260 aminoacid residues, 220-250 amino acid residues, 220-248 amino acid residues,220-246 amino acid residues, 220-244 amino acid residues, 225-280 aminoacid residues, 225-270 amino acid residues, 225-260 amino acid residues,225-250 amino acid residues, 225-248 amino acid residues, 225-246 aminoacid residues, 225-244 amino acid residues, 230-280 amino acid residues,230-270 amino acid residues, 230-260 amino acid residues, 230-250 aminoacid residues, 230-248 amino acid residues, 230-246 amino acid residues,230-244 amino acid residues, 235-280 amino acid residues, 235-270 aminoacid residues, 235-260 amino acid residues, 235-250 amino acid residues,235-248 amino acid residues, 235-246 amino acid residues, 235-244 aminoacid residues, 240-280 amino acid residues, 240-270 amino acid residues,240-260 amino acid residues, 240-250 amino acid residues, 240-248 aminoacid residues, 240-246 amino acid residues, 240-244 amino acid residues,242-280 amino acid residues, 242-270 amino acid residues, 242-260 aminoacid residues, 242-250 amino acid residues, 242-248 amino acid residues,242-246 amino acid residues, 242-244 amino acid residues, 243-280 aminoacid residues, 243-270 amino acid residues, 243-260 amino acid residues,243-250 amino acid residues, 243-248 amino acid residues, 243-246 aminoacid residues, 243-244 amino acid residues,

In still further embodiments, the oil is contacted:

with 0.5-200 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-200 mg enzyme protein (EP)/Kg oil of saidPC and PE-specific phospholipase C; such as

with 0.5-100 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-100 mg enzyme protein (EP)/Kg oil of saidPC and PE-specific phospholipase C;

with 0.5-25 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-25 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C;

with 0.5-15 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-15 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 0.5-10 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-10 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 0.5-5 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-5 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 1-200 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-200 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C; such as

with 1-100 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-100 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C;

with 1-25 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-25 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C;

with 1-15 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-15 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 1-10 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-10 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 1-5 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-5 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-200 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-200 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-100 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-100 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-50 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-50 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-25 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-25 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-15 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-15 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-10 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-10 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-7 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-7 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C, or

with 2-5 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-5 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C.

In a further embodiment, the PC and PE-specific phospholipase C is a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of: i) amino acid residues 34-278 of SEQ ID NO: 19 or aminoacid residues 1-246 of SEQ ID NO: 20; ii) amino acid residues 25-283 ofSEQ ID NO: 22 or amino acid residues 39-283 of SEQ ID NO: 22; iii) aminoacid residues 25-283 of SEQ ID NO: 24 or amino acid residues 39-283 ofSEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25; iv) aminoacid residues 25-283 of SEQ ID NO: 27 or amino acid residues 39-283 ofSEQ ID NO: 27; v) amino acid residues 28-289 of SEQ ID NO: 29 or aminoacid residues 52-289 of SEQ ID NO: 29 or amino acid residues 1-263 ofSEQ ID NO: 30; vi) amino acid residues 21-282 of SEQ ID NO: 32 or aminoacid residues 38-282 of SEQ ID NO: 32; vii) amino acid residues 25-280of SEQ ID NO: 38 or amino acid residues 36-280 of SEQ ID NO: 38; andvii) Purafine; or b) a polypeptide comprising an amino acid sequencewhich has at least 75% identity to one of the amino acid sequences ina); or c) a functional fragment of a) or b).

Another aspect of the present invention is a method for reducing thecontent of phospholipids in an oil composition, the method comprising:a) providing an oil composition containing a quantity of phospholipids,b) contacting said oil composition with a PC and PE-specificphospholipase C selected from i) a polypeptide comprising an amino acidsequence selected from the group consisting of: 1) amino acid residues34-278 of SEQ ID NO: 19 or amino acid residues 1-246 of SEQ ID NO: 20;2) amino acid residues 25-283 of SEQ ID NO: 24, amino acid residues39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of SEQ ID NO: 25;3) amino acid residues 25-283 of SEQ ID NO: 27 or amino acid residues39-283 of SEQ ID NO: 27; 4) amino acid residues 28-289 of SEQ ID NO: 29,or amino acid residues 52-289 of SEQ ID NO: 29 or amino acid residues1-263 of SEQ ID NO: 30; ii) a polypeptide comprising an amino acidsequence which has at least 75% identity to one of the amino acidsequences in i); or iii) a functional fragment of i) or ii) underconditions sufficient for the enzymes to react with the phospholipids tocreate diglyceride and phosphate ester, and c) separating the phosphateester from the oil composition.

Phospholipids are commonly measured in oil as “phosphorous content” inparts per million. Table 1 sets forth the typical amounts ofphospholipids present in the major oilseed crops, and the distributionof the various functional groups as a percentage of the phospholipidspresent in the oil.

TABLE 1 Typical levels and phospholipid distributions for commonoilseeds Soy Oil Canola Oil Sunflower Oil Phosphorous (ppm) 400-1500200-900 300-700 PC % 12-46  25-40 29-52 PE % 8-34 15-25 17-26 PA %17-26  10-20 15-30 PI % 2-15  2-25 11-22

The enzymes and processes of the invention can be used to achieve a morecomplete degumming of high phosphorous oils, e.g., an oil with more than200 ppm of phosphorous, preferably more than 300 ppm, 400 ppm, 500 ppm,600 ppm, 700 ppm, 800 ppm, 900 ppm, even more preferred the oil containsmore than 1000 ppm phosphorous.

Preferably the oil comprises phosphatidylcholine (PC),phosphatidylethanolamine (PE) and phosphatidyl inositol (PI). Preferablythe oil contains more than 50 ppm phosphorous originating fromphosphatidyl inositol (PI), more preferably it contains more than 75ppm, 100 ppm, 125 ppm PI, even more preferably it contains more than 150ppm, most preferably it contains more than 175 ppm phosphorousoriginating from PI. Preferably the oil contains more than 100 ppmphosphorous originating from phosphatidylcholine (PC), more preferablyit contains more than 150 ppm, 200 ppm, 250 ppm PC, even more preferablyit contains more than 300 ppm, most preferably it contains more than 400ppm phosphorous originating from PC. Preferably the oil contains morethan 75 ppm phosphorus originating from phosphatidylethanolamine (PE),more preferably it contains more than 100 ppm, 125 ppm, 150 ppm PE, evenmore preferably it contains more than 200 ppm, most preferably itcontains more than 300 ppm phosphorous originating from PE.

In a preferred embodiment the oil is an edible oil. More preferred theedible oil is selected from rice bran, rapeseeds, palm, peanuts andother nuts, soybean, corn, canola, and sunflower oils. Thephospholipases of the invention can be used in any “degumming”procedure, including water degumming, ALCON oil degumming (e.g., forsoybeans), safinco degumming, “super degumming,” UF degumming, TOPdegumming, uni-degumming, dry degumming and ENZYMAX™ degumming. See, forexample, WO 2007/103005, US 2008/0182322, U.S. Pat. Nos. 6,355,693,6,162,623, 6,103,505, 6,001,640, 5,558,781 and 5,264,367 for descriptionof degumming processes where phospholipases of the present invention canbe applied. Various “degumming” procedures incorporated by the methodsof the invention are described in Bockisch, M. (1998) In Fats and OilsHandbook, The extraction of Vegetable Oils (Chapter 5), 345-5 445, AOCSPress, Champaign, Ill. The phospholipases of the invention can be usedin the industrial application of enzymatic degumming of triglycerideoils as described, e.g., in EP 513 709. In a further embodiment the oilis selected from crude oil, water degummed oil, caustic refined oil andacid degummed oil. The water-degumming of a crude oil or fat may beachieved by thoroughly mixing hot water and warm oil or fat having atemperature of between 50° C. to 90° C. for 30 to 60 minutes. Thisprocess serves to partially remove the hydratable phospholipids. Also,an acid treatment may be performed before the enzymatic degumming, wherethe acid used is selected from the group consisting of phosphoric acid,acetic acid, citric acid, tartaric acid, succinic acid, and mixturesthereof, in particular, a treatment using citric acid or phosphoric acidis preferred. The acid treatment is preferably followed by aneutralization step to adjust the pH between about 4.0 to 7.0, morepreferably from 4.5 to 6.5, preferably using NaOH or KOH. The acidtreatment serves to chelate metals bound to the phospholipids herebymaking a more hydratable form. Preferably, the phospholipases asdescribed herein is added after water degumming or acid treatment of theoil. It is also possible to perform the degumming step using thephospholipases as described herein on a crude oil or fat, i.e., an oilor fat not previously water degummed or acid treated.

In one aspect, the invention provides methods for enzymatic degummingunder conditions of low water, e.g., in the range of between about 0.1%to 20% water or 0.5% to 10% water. In one aspect, this results in theimproved separation of a heavy phase from the oil phase duringcentrifugation. The improved separation of these phases can result inmore efficient removal of phospholipids from the oil, including bothhydratable and nonhydratable phospholipids. In one aspect, this canproduce a gum fraction that contains less entrained neutral oil(triglycerides), thereby improving the overall yield of oil during thedegumming process. In one aspect, phospholipases of the invention, e.g.,a PI-specific-PLC and/or PC, PE-specific PLC, are used to treat oils toreduce gum mass and increase neutral oil gain through reduced oilentrapment. In one aspect, phospholipases of the invention, e.g., apolypeptide having PLC activity, are used for diacylglycerol (DAG)production and to contribute to the oil phase.

The phospholipase treatment can be conducted by dispersing an aqueoussolution of the phospholipase, preferably as droplets with an averagediameter below 10 μM. The amount of water is preferably 0.5-5% by weightin relation to the oil. An emulsifier may optionally be added.Mechanical agitation may be applied to maintain the emulsion. Agitationmay be done with a high shear mixer with a tip speed above 1400 cm/s.

In certain embodiments, a suitable oil degumming method comprises a)mixing an aqueous acid with an oil to obtain an acidic mixture having pHof about 1 to 4, b) mixing a base with the acidic mixture to obtain areacted mixture having pH of about 6-9, and c) degumming the reactedmixture with an enzyme of the present invention to obtain a degummedoil. In certain embodiments, mixing in steps a) and/or b) creates anemulsion that comprises an aqueous phase in average droplet size betweenabout 15 μM to about 45 μM. In certain embodiments, mixing in steps a)and/or b) creates an emulsion that comprises at least about 60% of anaqueous phase by volume in droplet size between about 15 μM to about 45μM in size, wherein percentage of the aqueous phase is based on thetotal volume of the aqueous phase. Any acid deemed suitable by one ofskill in the art can be used in the methods provided herein. In certainembodiments, the acid is selected from the group consisting ofphosphoric acid, acetic acid, citric acid, tartaric acid, succinic acid,and a mixture thereof. Any acid deemed suitable by one of skill in theart can be used in the methods provided herein. In certain embodiments,the base is selected from the group consisting of sodium hydroxide,potassium hydroxide, sodium silicate, sodium carbonate, calciumcarbonate, and a combination thereof.

In a preferred embodiment, the phospholipase treatment can be conductedat a pH in the range of about 4.0 to 7.0, more preferably from 4.5 to6.5. The pH is measured in the emulsion or in the interphase between thebetween oil and aqueous solution. A suitable temperature is generally30-80° C. In a preferred embodiment the temperature of the oil isbetween 50 and 70° C., more preferred between 55 and 65° C. and mostpreferred between 50 and 60° C. In other preferred embodiments thetemperature of the oil is between 60 and 80° C., more preferred between65 and 75° C. and most preferred between 67 and 72° C.

The reaction time will typically be 1-12 hours (e.g., 1-6 hours, or 1-3hours, most preferred the reaction time is between 1.5 and 4 hours, evenmore preferred between 1.5 and 2 hours). A suitable enzyme dosage willusually be 0.1-10 mg per liter (e.g., 0.5-5 mg per liter). Thephospholipase treatment may be conducted batch wise, e.g., in a tankwith stirring, or it may be continuous, e.g., a series of stirred tankreactors. The phospholipase treatment may be followed by separation ofan aqueous phase and an oil phase. The separation may be performed byconventional means, e.g., centrifugation. When a liquid lipase is usedthe aqueous phase will contain phospholipase, and the enzyme may bere-used to improve the process economy.

In a preferred embodiment of the present invention, the treatmentreduces the total phosphorous content of the oil to below 200 ppm,preferably below 100 ppm, below 50 ppm, below 40 ppm, 30 ppm, 20 ppm, 15ppm, more preferably below 10 ppm, below 9 ppm, below 8 ppm, below 7ppm, below 6 ppm, most preferably below 5 ppm.

In addition to the phospholipases of the present invention, a furtherenzyme may be applied in the degumming process outlined above. In apreferred embodiment the further enzyme is a polypeptide havingphospholipase A1, A2 and/or B activity. A suitable polypeptide havingphospholipase A1 activity may be LECITASE ULTRA available from NovozymesA/S.

Phospholipid emulsifiers: The phospholipase of the invention may be usedfor partial hydrolysis of phospholipids, preferably lecithin, to obtainimproved phospholipid emulsifiers. This application is further describedin Ullmann's Encyclopedia of Industrial Chemistry (Publisher: VCHWeinheim (1996)), JP 2794574, and JP-B 6-087751.

Filtration: The phospholipase of the invention can be used to improvethe filterability of an aqueous solution or slurry of carbohydrateorigin by treating it with the phospholipase. This is particularlyapplicable to a solution of slurry containing a starch hydrolyzate,especially a wheat starch hydrolyzate, since this tends to be difficultto filter and to give cloudy filtrates. The treatment can be done inanalogy with EP 219269 (CPC International).

Animal feed: The phospholipase of the invention may be used in a processfor the production of an animal feed which comprises mixing thephospholipase with feed substances comprising at least one phospholipid.This can be done in analogy with EP 743017.

Biodiesel: The phospholipase of the present invention may be used incombination with one or more lipolytic enzymes to convert fats and oilsto fatty acid alkyl esters while achieving degumming in the sameprocess. Such a process is for example described in U.S. Pat. No.8,012,724.

Detergent: The phospholipase of the invention may be added to and thusbe used as a component of a detergent composition.

The detergent composition may for example be formulated as a hand ormachine laundry detergent composition including a laundry additivecomposition suitable for pretreatment of stained fabrics and a rinseadded fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

Baking: The phospholipase of the invention may be used for production ofdough and baked products from dough, as well as for production of bakingcompositions and baking additives.

The dough generally comprises wheat meal or wheat flour and/or othertypes of meal, flour or starch such as corn flour, corn starch, ryemeal, rye flour, oat flour, oat meal, soy flour, sorghum meal, sorghumflour, potato meal, potato flour or potato starch.

The dough may be fresh, frozen or par-baked.

The dough is normally leavened dough or dough to be subjected toleavening. The dough may be leavened in various ways, such as by addingchemical leavening agents, e.g., sodium bicarbonate or by adding aleaven (fermenting dough), but it is preferred to leaven the dough byadding a suitable yeast culture, such as a culture of Saccharomycescerevisiae (baker's yeast), e.g., a commercially available strain of S.cerevisiae.

The dough may also comprise other conventional dough ingredients, e.g.,proteins, such as milk powder, gluten, and soy; eggs (either whole eggs,egg yolks or egg whites); an oxidant such as ascorbic acid, potassiumbromate, potassium iodate, azodicarbonamide (ADA) or ammoniumpersulfate; an amino acid such as L-cysteine; a sugar; a salt such assodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

The dough may comprise fat (triglyceride) such as granulated fat orshortening, but the invention is particularly applicable to a doughwhere less than 1% by weight of fat is added, and particularly to adough which is made without addition of fat.

The dough may further comprise an emulsifier such as mono- ordiglycerides, diacetyl tartaric acid esters of mono- or diglycerides,sugar esters of fatty acids, polyglycerol esters of fatty acids, lacticacid esters of monoglycerides, acetic acid esters of monoglycerides,polyoxyethylene stearates, or lysolecithin.

The dough may be used for any kind of baked product prepared from dough,either of a soft or a crisp character, either of a white, light or darktype. Examples are bread (in particular, white, whole-meal or ryebread), typically in the form of loaves or rolls, French baguette-typebread, pita bread, tortillas, cakes, pancakes, biscuits, wafers,cookies, pie crusts, crisp bread, steamed bread, pizza and the like.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

Items

1. A method for reducing the content of phospholipids in an oilcomposition, the method comprising

a) providing an oil composition containing a quantity of phospholipids,

b) contacting said oil composition with a phosphatidylinositolphospholipase C and a PC and PE-specific phospholipase C underconditions sufficient for the enzymes to react with the phospholipids tocreate diglyceride and phosphate ester; and

c) separating the phosphate ester from the oil composition.

2. The method of item 1 wherein said phosphatidylinositol phospholipaseC is from the genus of Pseudomonas.3. The method of item 1 or 2, wherein the oil is an edible oil.4. The method of any of the preceding items, wherein the oil is selectedfrom group consisting of crude oil, water degummed oil, caustic refinedoil and acid degummed oil.5. The method of any of the proceeding items, where in the oil comprisesphosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI).6. The method of item 5, wherein the oil comprises at least 50 ppmphosphorus originating from phosphatidyl inositol (PI).7. The method of any of the proceeding items, where thephosphatidylinositol phospholipase C is

a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   i) amino acid residues 26-322 of SEQ ID NO: 2 or amino acid        residues 1-298 of SEQ ID NO: 3;    -   ii) amino acid residues 26-323 of SEQ ID NO: 5 or amino acid        residues 1-299 of SEQ ID NO: 6;    -   iii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acid        residues 1-299 of SEQ ID NO: 9;    -   iv) amino acid residues 26-323 of SEQ ID NO: 11 or amino acid        residues 1-296 of SEQ ID NO: 12;    -   v) amino acid residues 26-322 of SEQ ID NO: 14 or amino acid        residues 1-298 of SEQ ID NO: 15;    -   vi) amino acid residues 26-322 of SEQ ID NO: 17; and    -   vii) amino acid residues 28-339 of SEQ ID NO: 35; or

b) a polypeptide comprising an amino acid sequence which has at least75% identity to one of the amino acid sequences in a); or

c) a functional fragment of a) or b).

8. The method of any of the proceeding items, where the PC andPE-specific phospholipase C is

a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid        residues 1-246 of SEQ ID NO: 20;    -   ii) amino acid residues 25-283 of SEQ ID NO: 22 or amino acid        residues 39-283 of SEQ ID NO: 22;    -   iii) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid        residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of        SEQ ID NO: 25;    -   iv) amino acid residues 25-283 of SEQ ID NO: 27 or amino acid        residues 39-283 of SEQ ID NO: 27;    -   v) amino acid residues 28-289 of SEQ ID NO: 29 or amino acid        residues 52-289 of SEQ ID NO: 29 or amino acid residues 1-263 of        SEQ ID NO: 30;    -   vi) amino acid residues 21-282 of SEQ ID NO: 32 or amino acid        residues 38-282 of SEQ ID NO: 32;    -   vii) amino acid residues 25-280 of SEQ ID NO: 38 or amino acid        residues 36-280 of SEQ ID NO: 38; and    -   viii) Purifine; or

b) a polypeptide comprising an amino acid sequence which has at least75% identity to one of the amino acid sequences in a); or

c) a functional fragment of a) or b).

9. The method of any of the preceding items, wherein saidphosphatidylinositol phospholipase C is a polypeptide having a length of280-320 amino acid residues, such as a length of 280-310 amino acidresidues, 280-305 amino acid residues, 280-300 amino acid residues,280-298 amino acid residues, 280-297 amino acid residues, 280-296 aminoacid residues, 285-320 amino acid residues, 285-315 amino acid residues,285-310 amino acid residues, 285-305 amino acid residues, 285-300 aminoacid residues, 285-298 amino acid residues, 285-297 amino acid residues,285-296 amino acid residues, 290-320 amino acid residues, 290-315 aminoacid residues, 290-310 amino acid residues, 290-305 amino acid residues,290-300 amino acid residues, 290-298 amino acid residues, 290-297 aminoacid residues, 290-296 amino acid residues, 295-320 amino acid residues,295-315 amino acid residues, 295-310 amino acid residues, 295-305 aminoacid residues, 295-300 amino acid residues, 295-298 amino acid residues,255-297 amino acid residues, or a length of 295-296 amino acid residues.10. The polypeptide of any of the preceding items, wherein said PC andPE-specific phospholipase C is a polypeptide having a length of 220-280amino acid residues, such as a length of 220-270 amino acid residues,220-260 amino acid residues, 220-250 amino acid residues, 220-248 aminoacid residues, 220-246 amino acid residues, 220-244 amino acid residues,225-280 amino acid residues, 225-270 amino acid residues, 225-260 aminoacid residues, 225-250 amino acid residues, 225-248 amino acid residues,225-246 amino acid residues, 225-244 amino acid residues, 230-280 aminoacid residues, 230-270 amino acid residues, 230-260 amino acid residues,230-250 amino acid residues, 230-248 amino acid residues, 230-246 aminoacid residues, 230-244 amino acid residues, 235-280 amino acid residues,235-270 amino acid residues, 235-260 amino acid residues, 235-250 aminoacid residues, 235-248 amino acid residues, 235-246 amino acid residues,235-244 amino acid residues, 240-280 amino acid residues, 240-270 aminoacid residues, 240-260 amino acid residues, 240-250 amino acid residues,240-248 amino acid residues, 240-246 amino acid residues, 240-244 aminoacid residues, 242-280 amino acid residues, 242-270 amino acid residues,242-260 amino acid residues, 242-250 amino acid residues, 242-248 aminoacid residues, 242-246 amino acid residues, 242-244 amino acid residues,243-280 amino acid residues, 243-270 amino acid residues, 243-260 aminoacid residues, 243-250 amino acid residues, 243-248 amino acid residues,243-246 amino acid residues, 243-244 amino acid residues.11. The method of any of the preceding items, wherein said oil iscontacted:

with 0.5-200 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-200 mg enzyme protein (EP)/Kg oil of saidPC and PE-specific phospholipase C; such as

with 0.5-100 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-100 mg enzyme protein (EP)/Kg oil of saidPC and PE-specific phospholipase C;

with 0.5-25 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-25 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C;

with 0.5-15 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-15 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 0.5-10 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-10 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 0.5-5 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 0.5-5 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 1-200 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-200 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C; such as

with 1-100 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-100 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C;

with 1-25 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-25 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C;

with 1-15 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-15 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 1-10 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-10 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 1-5 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 1-5 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-200 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-200 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-100 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-100 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-50 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-50 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-25 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-25 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-15 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-15 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-10 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-10 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-7 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-7 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C,

with 2-5 mg enzyme protein (EP)/Kg oil of said phosphatidylinositolphospholipase C and with 2-5 mg enzyme protein (EP)/Kg oil of said PCand PE-specific phospholipase C.

12. A polypeptide having phosphatidylinositol phospholipase C activity,selected from the group consisting of:

a) a polypeptide having at least 91% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

b) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions with

-   -   i) the mature polypeptide coding sequence of SEQ ID NO: 1, or    -   ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more positions; and

e) a fragment of the polypeptide of (a), (b), (c), or (d) that hasphosphatidylinositol phospholipase C activity.

13. The polypeptide of item 12, said polypeptide having a thermaldenaturation temperature of at least 60° C., such as 61° C., 62° C., 63°C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72°C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81°C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C. or atleast 90° C. as determined by Differential Scanning calorimetry (DSC).14. The polypeptide of item 12 or 13, wherein said denaturationtemperature is determined as the top of denaturation peak (majorendothermic peak) in thermograms (Cp vs. T) obtained after heating a 1mg/ml solution of the polypeptide in buffer (50 mM Na-acetate pH 5.5, or50 mM Hepes pH 7) at a constant programmed heating rate of 200 K/hr.15. The polypeptide of any of items 12-14, said polypeptide being ableto reduce the phosphatidylinositol content of crude soy bean oil by 50%or more, 50%, such as by 55%, by 60%, by 65%, by 70%, by 75%, by 80%, by85%, by 90% or by 95% or more, the reduction in phosphatidylinositolcontent being determined by ³¹P-NMR after addition of 100 mg enzymeprotein (EP)/kg oil and incubation of the oil and enzyme at 50° C. for 2hours at pH 5.5.16. The polypeptide of any of items 12-15, said polypeptide being ableto reduce the phosphorous content of crude soy bean oil to 20 mg/kg oilor less as determined by Inductively coupled plasma optical emissionspectrometry (ICP-OES) after incubation of 4 mg enzyme protein/kg oil ina low aqueous system comprising 3% water based on oil amount at 50-60°C. for 5 hours.17. The polypeptide of item 16, wherein said crude soy oil comprises80-140 ppm phosphorous present as phosphatidic acid (PA), 140-200 ppmphosphorous present as phosphatidyl ethanolamine (PE), 70-110 ppmphosphorous present as phosphatidic acid (PI) and 130-200 ppmphosphorous present as phosphatidyl choline; the phosphorous contentbeing measured by ³¹P-NMR.18. The polypeptide of item 16 or 17, wherein the reduction ofphosphorous content is obtained in an oil degumming process comprisingthe steps of:

i) Optionally treating crude soy bean oil with acid/base by adding an85% solution of Ortho Phosphoric acid in amounts corresponding to 0.05%(100% pure Ortho Phosphoric acid) based on oil amount, mixing inultrasonic bath for 5 minutes, followed by incubation in rotator for 15minutes and base neutralization with 4 M NaOH applied in equivalents(from 0.5 to 0.15) to pure Ortho Phosphoric acid in ultrasonic bath for5 minutes;

ii) Adding the polypeptide to the oil in amounts of 4 mg enzymeprotein/kg oil in a low aqueous system comprising 3% water based on oilamount and subjecting the oil and the polypeptide to ultrasonictreatment for 5 minutes;

iii) Incubating the polypeptide and oil at 50-60° C. for 5 hours withstirring at 20 rpm;

iv) Centrifuging the oil and the polypeptide at 700 g at 85° C. for 15minutes.

19. The polypeptide of any of items 12-18, comprising, consisting of orconsisting essentially of SEQ ID NO: 2 or the mature polypeptide of SEQID NO: 2 or amino acids 26 to 322 of SEQ ID NO: 2 or to amino acids 1 to298 of SEQ ID NO: 3.20. The polypeptide of any of items 12-19, having a length of 280-320amino acid residues, such as a length of 280-310 amino acid residues,280-305 amino acid residues, 280-300 amino acid residues, 280-298 aminoacid residues, 280-297 amino acid residues, 280-296 amino acid residues,285-320 amino acid residues, 285-315 amino acid residues, 285-310 aminoacid residues, 285-305 amino acid residues, 285-300 amino acid residues,285-298 amino acid residues, 285-297 amino acid residues, 285-296 aminoacid residues, 290-320 amino acid residues, 290-315 amino acid residues,290-310 amino acid residues, 290-305 amino acid residues, 290-300 aminoacid residues, 290-298 amino acid residues, 290-297 amino acid residues,290-296 amino acid residues, 295-320 amino acid residues, 295-315 aminoacid residues, 295-310 amino acid residues, 295-305 amino acid residues,295-300 amino acid residues, 295-298 amino acid residues, 255-297 aminoacid residues, or a length of 295-296 amino acid residues.21. A polypeptide having PC and PE specific phospholipase C activity,selected from the group consisting of:

a) a polypeptide having at least 70% sequence identity to the maturepolypeptide of SEQ ID NO: 19;

b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with

-   -   i) the mature polypeptide coding sequence of SEQ ID NO: 18, or    -   ii) the full-length complement of (i);

c) a polypeptide encoded by a polynucleotide having at least 70%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 18;

d) a variant of the mature polypeptide of SEQ ID NO: 19 comprising asubstitution, deletion, and/or insertion at one or more positions; and

e) a fragment of the polypeptide of (a), (b), (c), or (d) that has PCand PE specific phospholipase C activity.

22. The polypeptide of item 21, said polypeptide having a thermaldenaturation temperature of at least 60° C., such as 61° C., 62° C., 63°C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72°C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81°C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C. or atleast 90° C. as determined by Differential Scanning calorimetry (DSC).23. The polypeptide of item 22, wherein said denaturation temperature isdetermined as the top of denaturation peak (major endothermic peak) inthermograms (Cp vs. T) obtained after heating a 1 mg/ml solution of thepolypeptide in buffer (50 mM Na-acetate pH 5.5, or 50 mM Hepes pH 7) ata constant programmed heating rate of 200 K/hr.24. The polypeptide of any of items 21-23, said polypeptide being ableto reduce the phosphatidyl ethanolamine and/or phosphatidyl cholinecontent of crude soy bean oil by 50% or more, 50%, such as by 55%, by60%, by 65%, by 70%, by 75%, by 80%, by 85%, by 90% or by 95% or more,the reduction in phosphatidyl ethanolamine and/or phosphatidyl cholinecontent being determined by ³¹P-NMR after addition of 100 mg enzymeprotein (EP)/kg oil and incubation of the oil and enzyme at 50° C. for 2hours at pH 5.5.25. The polypeptide of any of items 21-24, said polypeptide being ableto reduce the phosphorous content of crude soy bean oil to 20 mg/kg oilor less as determined by Inductively coupled plasma optical emissionspectrometry (ICP-OES) after incubation of 4 mg enzyme protein/kg oil ina low aqueous system comprising 3% water based on oil amount at 50-60°C. for 5 hours.26. The polypeptide of item 24 or 25, wherein said crude soy oilcomprises 80-140 ppm phosphorous present as phosphatidic acid (PA),140-200 ppm phosphorous present as phosphatidyl ethanolamine (PE),70-110 ppm phosphorous present as phosphatidic acid (PI) and 130-200 ppmphosphorous present as phosphatidyl choline; the phosphorous contentbeing measured by ³¹P-NMR.

27. The polypeptide of any of items 24-26, wherein the reduction ofphosphorous content and/or reduction in phosphatidyl ethanolamine and/orphosphatidyl choline content is obtained in an oil degumming processcomprising the steps of:

i) Optionally treating crude soy bean oil with acid/base by adding an85% solution of Ortho Phosphoric acid in amounts corresponding to 0.05%(100% pure Ortho Phosphoric acid) based on oil amount, mixing inultrasonic bath for 5 minutes, followed by incubation in rotator for 15minutes and base neutralization with 4 M NaOH applied in equivalents(from 0.5 to 0.15) to pure Ortho Phosphoric acid in ultrasonic bath for5 minutes;

ii) Adding the polypeptide to the oil in amounts of 4 mg enzymeprotein/kg oil in a low aqueous system comprising 3% water based on oilamount and subjecting the oil and the polypeptide to ultrasonictreatment for 5 minutes;

iii) Incubating the polypeptide and oil at 50-60° C. for 5 hours withstirring at 20 rpm;

iv) Centrifuging the oil and the polypeptide at 700 g at 85° C. for 15minutes.

28. The polypeptide of any of items 21-27, comprising, consisting of orconsisting essentially of SEQ ID NO: 19 or the mature polypeptide of SEQID NO: 19 or amino acids 34 to 278 of SEQ ID NO: 19 or amino acidresidues 1-246 of SEQ ID NO: 20.29. The polypeptide of any of items 21-28, having a length of 220-280amino acid residues, such as a length of 220-270 amino acid residues,220-260 amino acid residues, 220-250 amino acid residues, 220-248 aminoacid residues, 220-246 amino acid residues, 220-244 amino acid residues,225-280 amino acid residues, 225-270 amino acid residues, 225-260 aminoacid residues, 225-250 amino acid residues, 225-248 amino acid residues,225-246 amino acid residues, 225-244 amino acid residues, 230-280 aminoacid residues, 230-270 amino acid residues, 230-260 amino acid residues,230-250 amino acid residues, 230-248 amino acid residues, 230-246 aminoacid residues, 230-244 amino acid residues, 235-280 amino acid residues,235-270 amino acid residues, 235-260 amino acid residues, 235-250 aminoacid residues, 235-248 amino acid residues, 235-246 amino acid residues,235-244 amino acid residues, 240-280 amino acid residues, 240-270 aminoacid residues, 240-260 amino acid residues, 240-250 amino acid residues,240-248 amino acid residues, 240-246 amino acid residues, 240-244 aminoacid residues, 242-280 amino acid residues, 242-270 amino acid residues,242-260 amino acid residues, 242-250 amino acid residues, 242-248 aminoacid residues, 242-246 amino acid residues, 242-244 amino acid residues,243-280 amino acid residues, 243-270 amino acid residues, 243-260 aminoacid residues, 243-250 amino acid residues, 243-248 amino acid residues,243-246 amino acid residues, 243-244 amino acid residues.30. A polynucleotide encoding a polypeptide of any of items 12 to 29.31. A nucleic acid construct or expression vector comprising thepolynucleotide of item 30 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.32. A recombinant host cell comprising the polynucleotide of item 30operably linked to one or more control sequences that direct theproduction of the polypeptide.33. A method of producing a polypeptide of any of items 12 to 29,comprising cultivating a cell, which in its wild-type form produces thepolypeptide, under conditions conducive for production of thepolypeptide.34. A method of producing a phospholipase C polypeptide in a Bacillushost, wherein the phospholipase C coding sequence encode an alanine infront of the predicted N-terminal amino acid of the phospholipase Cpolypeptide.35. The method of item 34, wherein the phospholipase C polypeptide is aPC and PE-specific phospholipase C polypeptide.36. A method of producing a polypeptide of any of items 12 to 29,comprising cultivating the host cell of item 32 under conditionsconducive for production of the polypeptide.37. The method of any of items 33-37, further comprising recovering thepolypeptide.38. A composition comprising the polypeptide of any of items 12-20.39. A composition comprising the polypeptide of any of items 21-29.40. A composition comprising a mixture of a phosphatidylinositolphospholipase C from the genus of Pseudomonas and a PC and PE-specificphospholipase C polypeptide.41. The composition of item 40, wherein the phosphatidylinositolphospholipase C is:

a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   i) amino acid residues 26-322 of SEQ ID NO: 2 or amino acid        residues 1-298 of SEQ ID NO: 3;    -   ii) amino acid residues 26-323 of SEQ ID NO: 5 or amino acid        residues 1-299 of SEQ ID NO: 6;    -   iii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acid        residues 1-299 of SEQ ID NO: 9;    -   iv) amino acid residues 26-323 of SEQ ID NO: 11 or amino acid        residues 1-296 of SEQ ID NO: 12;    -   v) amino acid residues 26-322 of SEQ ID NO: 14 or amino acid        residues 1-298 of SEQ ID NO: 15;    -   vi) amino acid residues 26-322 of SEQ ID NO: 17; and    -   vii) amino acid residues 28-339 of SEQ ID NO: 35; or

b) a polypeptide comprising an amino acid sequence which has at least75% identity to one of the amino acid sequences in a); or

c) a functional fragment of a) or b).

42. The composition of item 41, wherein the phosphatidylinositolphospholipase C is the polypeptide of any of items 12-20.43. The composition of any of items 38-42, wherein the PC andPE-specific phospholipase C polypeptide is:

a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   i) amino acid residues 34-278 of SEQ ID NO: 19 or amino acid        residues 1-246 of SEQ ID NO: 20;    -   ii) amino acid residues 25-283 of SEQ ID NO: 22 or amino acid        residues 39-283 of SEQ ID NO: 22;    -   iii) amino acid residues 25-283 of SEQ ID NO: 24 or amino acid        residues 39-283 of SEQ ID NO: 24 or amino acid residues 1-260 of        SEQ ID NO: 25;    -   iv) amino acid residues 39-283 of SEQ ID NO: 27;    -   v) amino acid residues 52-289 of SEQ ID NO: 29 or amino acid        residues 1-263 of SEQ ID NO: 30    -   vi) amino acid residues 21-282 of SEQ ID NO: 32 or amino acid        residues 38-282 of SEQ ID NO: 32;    -   vii) amino acid residues 25-280 of SEQ ID NO: 38 or amino acid        residues 36-280 of SEQ ID NO: 38; and    -   viii) Purafine; or

b) a polypeptide comprising an amino acid sequence which has at least75% identity to one of the amino acid sequences in a); or

c) a functional fragment of a) or b).

EXAMPLES Strains and DNA

The DNA encoding the PLC of SEQ ID NO: 2 was cloned from a Pseudomonasspecies isolated from seaweed sample collected in Denmark.

The DNA encoding the PLC of SEQ ID NO: 19 was cloned from a Bacillus sp.obtained from a soil sample collected in Australia in 1990.

The codon optimized DNA encoding the publicly known PLC's was orderedfrom the companies Geneart (SEQ ID NO: 22, SEQ ID NO: 27 and SEQ ID NO:29) and Gen9 (SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14).

In the examples below the phospholipase C enzymes of the presentinvention are referred to by SEQ ID NO. If the SEQ ID NO contains asignal peptide it is understood that the reference is to the maturesequence of that SEQ ID NO.

Example 1: Cloning and Expression

The phospholipase encoding genes were either cloned by conventionaltechniques from the strains indicated above or ordered as syntheticgenes and inserted into a suitable plasmid. The genes were expressedwith the secretion signal having the following amino acid sequenceMKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO: 33) replacing the nativesecretion signal sequence with an extra alanine at the C-terminal. Thisresults in a recombinant mature polypeptide with an alanine at the frontof the N-terminal of the mature wild type sequence. Genes encoding SEQID NO: 22, 27 and SEQ ID NO: 38 were cloned using the same strategy,however, no extra alanine was added at the C-terminal of the signalpeptide. Thus, the recombinant mature polypeptide does not contain analanine at the front of the N-terminal of the mature wild type sequence.

One clone with the correct recombinant gene sequence was selected andthe corresponding plasmid was integrated by homologous recombinationinto the Bacillus subtilis host cell genome (pectate lyase locus) andthe gene construct was expressed under the control of a triple promotersystem as described in WO 99/43835. The gene coding for chloramphenicolacetyltransferase was used as a marker (as described in Diderichsen etal., 1993, Plasmid 30:312-315).

Chloramphenicol resistant transformants were analyzed by PCR to verifythe correct size of the amplified fragment. A recombinant B. subtilisclone containing the integrated expression construct was selected and itwas cultivated on a rotary shaking table in 500 mL baffled Erlenmeyerflasks each containing 100 ml yeast extract-based media. The clone wascultivated for 5 days at 30° C. The enzyme containing supernatants wereharvested and the enzyme purified as described in Example 2.

Example 2: Phospholipase C Purification Purification of the MaturePeptide of SEQ ID NO: 3:

The cell-free culture broth was buffer-exchanged to 50 mM MES pH 6.5using a packed bed of Sephadex® G-25 resin. The collected fractions wereloaded onto a Source 15S cation-exchanger and eluted using a gradient of0-100% 50 mM MES+0.5 M NaCl pH 6.5 in 10 CV's. Fractions were analyzedby SDS-PAGE (reducing conditions) and pooled based on purity.

Purification of the Mature Polypeptide of SEQ ID NO: 20:

Initially, an impurity capture step was performed using a packed bed ofa decylamine agarose (Acetyleret Decylaminagarose, Cat. no. CS76,UpFront Cromatography A/S, Lersø Parkalle 42, 2100 Copenhagen Ø,Denmark). Binding buffer: 25 mM HEPES pH 7.5. Elution buffer: Bindingbuffer+0.01% (w/v) Triton X-100. The flow-through and wash fractionswere pooled and buffer-exchanged to 50 mM MES pH 6.0 by loading onto apacked bed of Sephadex® G-25 resin. The collected fractions were loadedonto a Source 15Q anion-exchanger and eluted using a gradient of 0-100%50 mM MES+0.5 M NaCl pH 6.0 in 10 CV's. Fractions were analyzed bySDS-PAGE (reducing conditions) and pooled based on purity. The pooledfractions were concentrated by use of centrifugal filter devices w/10kDa MWCO and loaded onto a HiLoad™ 26/60 Superdex 200 pg gel filtrationcolumn equilibrated using 20 mM MES+125 mM NaCl pH 6.0. Fractions wereanalyzed by SDS-PAGE (reducing conditions) and pooled based on purity.

Example 3: Molecular Weight and N-Terminal Sequences of PLCDetermination of Molecular Weight:

The intact molecular weight analyses were performed using a BrukermicroTOF focus electrospray mass spectrometer (Bruker Daltonik GmbH,Bremen, DE). The samples were diluted to 1 mg/ml in MQ water. Thediluted samples were online washed on a MassPREP OnLine Desalting column(2.1×10 mm Part no. 186002785 Waters) and introduced to the electrospraysource with a flow of 200 ul/h by an Agilent LC system. Data analysis isperformed with DataAnalysis version 3.4 (Bruker Daltonik GmbH, Bremen,DE). The molecular weight of the samples was calculated by deconvolutionof the raw data in the range 10.000 to 40.000 Da.

The molecular weight of the PI-specific phospholipase of SEQ ID NO: 3was 32.7 kDa.

The molecular weight of the PC, PE-specific phospholipase of SEQ ID NO:20 was 27.6 kDa.

N-Terminal Sequencing Procedure:

N-terminal sequencing analyses were performed using an AppliedBiosystems Procise® protein sequencing system. The samples were purifiedon a Novex® precast 4-20% SDS polyacrylamide gel (Life Technologies).The gel was run according to manufacturer's instructions and blotted toa ProBlott® PVDF membrane (Applied Biosystems). For N-terminal aminoacid sequencing the main protein band was cut out and placed in theblotting cartridge of the Procise® protein sequencing system. TheN-terminal sequencing was carried out using the method run file for PVDFmembrane samples (Pulsed liquid PVDF) according to manufacturer'sinstructions. The N-terminal amino acid sequence can be deduced from the7 chromatograms corresponding to amino acid residues 1 to 7 by comparingthe retention time of the peaks in the chromatograms to the retentiontimes of the PTH-amino-acids in the standard chromatogram.

The N-terminal sequence of the mature polypeptide (SEQ ID NO: 3) wasconfirmed to be AQESPAF.

The N-terminal sequence of the mature polypeptide (SEQ ID NO: 6) wasconfirmed to be AQEAVGF.

The N-terminal sequence of the mature polypeptide (SEQ ID NO: 9) wasconfirmed to be AQEAVGF.

The N-terminal sequence of the mature polypeptide (SEQ ID NO: 20) wasconfirmed to be AWSADAP.

Example 4: Thermostability of the Phospholipase C

The thermostability of phospholipases C was determined by DifferentialScanning calorimetry (DSC) using a VP-Capillary Differential Scanningcalorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermaldenaturation temperature, Td (° C.), was taken as the top ofdenaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating enzyme solutions (approx. 1 mg/ml) in buffer (50mM Na-acetate pH 5.5±2 mM CaCl₂, or 50 mM Hepes pH 7±2 mM CaCl₂) at aconstant programmed heating rate of 200 K/hr.

Sample- and reference-solutions (approx. 0.2 ml) were loaded into thecalorimeter (reference: buffer without enzyme) from storage conditionsat 10° C. and thermally pre-equilibrated for 20 minutes at 20° C. priorto DSC scan from 20° C. to 100° C. Denaturation temperatures weredetermined at an accuracy of approximately +/−1° C.

TABLE 2 Denaturation temperatures Tm ° C. pH 5.5 Tm ° C. pH 7.0 SequenceID −CaCl2 +CaCl2 −CaCl2 +CaCl2 SEQ ID NO: 3 68 71 62.3 SEQ ID NO: 20 8483

Example 5: Phospholipase C Specificity Towards PC, PE, PI, PA ofPurified PLC's

The substrate specificity of the phospholipase C enzymes of the presentinvention and Purifine were determined using ³¹P-NMR. This assay followsthe conversion of individual phospholipids shown in FIG. 1 in an oilenvironment and reveals the substrate specificity and preference of thephospholipase, and provides an indication of the pH optimum of theenzymes.

Substrate

Crude soy bean oils with the following content of the specificphospholipids measured by P-NMR were used.

PA: 80-140 ppm Phosphorus (P) PE: 140-200 ppm P PI: 70-110 ppm P PC:130-200 ppm P

Other crude oils may also be applied in this assay, e.g., from rapeseed,sunflower, corn, cottonseed, groundnut, rice bran. The primary criterionis that the oil contains minimum 30 ppm of each of the specificphospholipids (to be significantly above the NMR quantification limit).Ensure mixing before the crude oil is pipetted (it precipitates overtime).

Buffers and Enzyme

0.2 M Cs-EDTA pH 7.5 solution: EDTA (5.85 g) is dispersed in MQ-water(50 mL). The pH is adjusted to 7.5 using 50% w/w CsOH (approx. 30 mL),which will dissolve the EDTA completely. MQ-water is added to a totalvolume of 100 mL to give a concentration of 0.2 M.

Internal standard: 2 mg/mL solution triphenyl phosphate (TPP) in MeOH.

pH buffers:

100 mM Na-citrate pH 4.0

100 mM Na-citrate pH 5.5

100 mM Na-citrate pH 7.0

Enzyme: Dilute to concentrations of 0.9, 0.27, and 0.09 mg EnzymeProtein (EP)/mL in the three buffers and keep cold to be used the sameday.

Assay

250 micro-L crude oil was weighed into a 2 mL Eppendorf and 25 micro-Lenzyme diluted in the desired pH buffer was added. This results in 10,30, and 100 mg EP/kg oil. The mixture was incubated in a thermoshaker at50° C. for 2 h. Then 0.500 mL phosphate standard solution, 0.5 mLchloroform-d (CDCl3) and 0.5 mL Cs-EDTA buffer was added. Phaseseparation was obtained after 30 seconds shaking followed bycentrifugation (tabletop centrifuge, 3 min, 13,400 rpm). The lower phasewas transferred to a NMR-tube. ³¹P NMR with 128 scans, 5 sec delay timewas run. All signals were integrated. Assignments (approx. ppm at 25°C.): 1.7 (PA), −0.1 (PE), −0.5 (PI), −0.8 (PC). The position of thesignals can change significantly according to exact pH value,temperature, sample concentration, etc. The concentration of eachspecies is calculated as “ppm P”, i.e., mg elemental Phosphorus per kgoil sample. Hence, ppm P=I/I(IS)*n(IS)*M(P)/m(oil). % Remainingphospholipid is calculated as the ratio of the phospholipidconcentration in the enzyme treated sample to the same concentration ina blank sample.

Results

The results are summarized in Tables 3 to 10 below.

TABLE 3 Specificity of phospholipase of SEQ ID NO: 3 10 mg EP/kg Oil 100mg EP/kg oil % Remaining phospholipid % Remaining phospholipid pH 4.0 pH5.5 pH 7.0 pH 4.0 pH 5.5 pH 7.0 PA 106 97 110 106 103 106 PE 103 94 9797 97 88 PI 88 44 20 52 12 12 PC 105 100 113 97 97 100

TABLE 4 Specificity of phospholipase of SEQ ID NO: 20. 10 mg EP/kg Oil100 mg EP/kg oil % Remaining phospholipid % Remaining phospholipid pH4.0 pH 5.5 pH 7.0 pH 4.0 pH 5.5 pH 7.0 PA 100 103 97 106 100 109 PE 10881 72 94 33 19 PI 100 96 100 88 100 96 PC 91 81 58 86 30 12

TABLE 5 Specificity of phospholipase of SEQ ID NO: 22 (amino acids25-283). 10 mg EP/kg Oil 100 mg EP/kg oil % Remaining phospholipid %Remaining phospholipid pH 4.0 pH 5.5 pH 7.0 pH 4.0 pH 5.5 pH 7.0 PA 10595 103 n.d. n.d. 61 PE 103 90 23 n.d. n.d. 0 PI 96 99 96 n.d. n.d. 90 PC98 61 6 n.d. n.d. 0

TABLE 6 Specificity of phospholipase of SEQ ID NO: 27. 10 mg EP/kg Oil100 mg EP/kg oil % Remaining phospholipid % Remaining phospholipid pH4.0 pH 5.5 pH 7.0 pH 4.0 pH 5.5 pH 7.0 PA 102 98 104 n.d. n.d. n.d. PE100 62 14 n.d. n.d. n.d. PI 97 99 95 n.d. n.d. n.d. PC 88 38 0 n.d. n.d.n.d.

TABLE 7 Specificity of phospholipase of SEQ ID NO: 30. 10 mg EP/kg Oil100 mg EP/kg oil % Remaining phospholipid % Remaining phospholipid pH4.0 pH 5.5 pH 7.0 pH 4.0 pH 5.5 pH 7.0 PA 107 106 105 102 86 71 PE 11796 70 102 29 0 PI 94 91 91 87 86 82 PC 112 97 48 108 23 11

TABLE 8 Specificity of phospholipase of SEQ ID NO: 36. 10 mg EP/kg Oil100 mg EP/kg oil % Remaining phospholipid % Remaining phospholipid pH4.0 pH 5.5 pH 7.0 pH 4.0 pH 5.5 pH 7.0 PA 110 111 115 n.d. n.d. n.d. PE109 113 109 n.d. n.d. n.d. PI 88 0 0 n.d. n.d. n.d. PC 112 109 102 n.d.n.d. n.d.

TABLE 9 Specificity of phospholipase of SEQ ID NO: 38 (amino acids36-280). 10 mg EP/kg Oil 100 mg EP/kg oil % Remaining phospholipid %Remaining phospholipid pH 4.0 pH 5.5 pH 7.0 pH 4.0 pH 5.5 pH 7.0 PA 8765 74 87 17 26 PE 109 0 7 70 0 0 PI 104 83 104 87 83 100 PC 98 78 4 82 00

TABLE 10 For an activity comparison, the performance of Purifine isshown below. Specificity of Purifine. 10 mg EP/kg Oil 100 mg EP/kg oil %Remaining phospholipid % Remaining phospholipid pH 4.0 pH 5.5 pH 7.0 pH4.0 pH 5.5 pH 7.0 PA 106 101 112 107 107 89 PE 106 114 18 107 38 0 PI 98106 102 91 106 94 PC 105 98 0 114 20 0

Purifine concentration is estimated to 15 mg/mL.

Example 6: Phospholipase C Specificity Towards PC, PE, PI, PA of CrudeEnzyme Supernatants

In addition to the results on purified enzyme samples in Example 5 thephospholipase activity of several PLC homologues were tested using crudeundiluted PLC supernatant in crude soy oil in a 1:10 (v/v) ratio. Theenzyme concentrations were unknown and there was no pH control,otherwise the protocol of Example 5 was followed.

TABLE 11 Phospholipid hydrolysis with crude undiluted PLC containingsupernatant. % Remaining phospholipid PA PE PI PC SEQ ID NO: 15 102 9914 105 SEQ ID NO: 12 124 109 54 109 SEQ ID NO: 9 113 99 27 111 SEQ IDNO: 6 114 109 49 107 SEQ ID NO: 25 101 10 87 0 SEQ ID NO: 27 96 8 100 3SEQ ID NO: 38 96 15 105 29 (amino acids 25-280) SEQ ID NO: 36 88 100 1792

Example 7: Degumming Assay

Performance of the phospholipase C enzymes of the present invention andPurifine as well as combinations of PI-specific and PC, PE specificphospholipase C enzymes were tested in a degumming assay that mimicsindustrial scale degumming. The assay measured the following parametersin the oil phase after the degumming:

a) Diglyceride content by High-performance liquid chromatography (HPLC)coupled to Evaporative Light Scattering Detector (ELSD), or ChargedAerosol Detector (Corona Veo).

b) Quantification of the individual phospholipids species:Phosphatidylcholine (PC); Phosphatidylinositol (PI);Phosphatidylethanolamine (PE); Phosphatidic acid (PA); by LiquidChromatography quadrupole mass spectrometer time of flight (LC/TOF/MS)

c) Total phosphorus reduction by inductively coupled plasma opticalemission spectrometry (ICP-OES).

The phospholipid composition in the crude soybean oil 2 or oil 3, usedin the experiments, is indicated in Table 12. The composition wasmeasured by LC/MS as phosphorus originating from individual phospholipidspecies.

TABLE 12 Phospholipid composition of crude oil (mg/kg phosphorus). Crudeoil 2 Crude oil 3 Crude oil 4- Crude oil 5 PA 295 85 <20 110 PE 125 187312 222 PI 84 182 85 64 PC 229 410 557 187 Total 732 864 974 583

Degumming Assay

Crude soybean oil (75 g) was initially acid/base pretreated (or not) tofacilitate conversion of insoluble phospholipids salt into morehydratable forms and ensure an environment suitable for the enzyme.Acid/base pretreatment was done by acid addition of Ortho Phosphoricacid (85% solution) applied in amounts equal to 0.05% (100% pure OrthoPhosphoric acid) based on oil amount and mixing in ultrasonic bath(BRANSON 3510) for 5 min and incubation in rotator for 15 min followedby base neutralization with 4 M NaOH applied in equivalents (from 0.5 to1.5) to pure Ortho Phosphoric acid in ultrasonic bath for 5 min. Theenzyme reaction was conducted in low aqueous system (3% water totalbased on oil amount) in 100 ml centrifuge tubes, cylindrical, conicalbottom. Samples were ultrasonic treated for 5 min, followed byincubation in a heated cabinet at selected temperature (from 50 to 60°C.) with stirring at 20 rpm for a selected incubation time (from 1 to 5hours). To separate the mixture into an oil phase and a heavy water/gumphase the samples were centrifuged at 700 g at 85° C. for 15 min(Koehler Instruments, K600×2 oil centrifuge).

a) Diglyceride Measurement

The HPLC-ELSD or HPLC-Corona Veo method (using DIONEX equipment andLichrocart Si-60, 5 μm, Lichrosphere 250-4 mm, MERCK column) was basedon the principle of the AOCS Official Method Cd 11d-96 and quantifiesthe diglyceride content down to 0.1 wt %.

b) Quantitative Analysis of Phospholipids by LCMS/MS

Liquid Chromatography coupled to triple quadrupole mass spectrometer(LC/MS/MS) or coupled to quadrupole mass spectrometer time of flight(LC/TOF/MS) was used to quantify the individual phospholipids species:phosphatidylcholine (PC); Phosphatidylinositol (PI);Phosphatidylethanolamine (PE) and Phosphatidic acid (phosphatidate)(PA). The sensitivity of the assay goes down to less than 1 mgPhosphorus/kg oil for PC, PE and PI (ppm) and less than 10 mgPhosphorus/kg for PA. The oil sample was dissolved in chloroform. Theextract was then analysed on LC-TOF-MS (or on LC-MS/MS if lowerdetection limits are needed) using the following settings:

LC-Settings

Eluent A: 50% Acetonitril, 50% Water, 0.15% formic acidEluent B: 100% Isopropionic acid, 0.15% formic acidRun time: 26.9 minFlow: 0.50 mL/minColumn temperature: 50° C.Autosampler temp: 15-25° C.Injection volume: 1 μLColumn type Material: Charged Surface Hybrid, length: 50 mm, size: 1.7μm, ID: 2.1 mm

MS-Settings

MS-settings TOF/MS MS/MS (Xevo) Capillary: 3.50 kV Capillary: +3.50/−2.0kV □□Cone: 28 Cone: Component specific □□Extractor: 2 V □□Extractor: 2.5V □□RF-lens: 0.5 V □□RF-lens: □□Source temp: 125° C. □□Source temp: 150°C. □□Desolvation temp: 500° C. □□Desolvation temp: 500° C. □□Cone gasflow: 30 L/hour □□Cone gas flow: 30 L/hour □□Desolvation gas flow:□□Desolvation gas flow: 850 L/hour 850 L/hour

The data was processed using MassLynx version 4.1 Software. In the belowexamples the method is just termed LCMS.

c) Phosphorus/Phospholipid Measurement

The ICP-OES quantifies the phosphorus (P) content and other metals suchas Ca, Mg, Zn down to 4 ppm with an accuracy of approximately ±1 ppm P.

Examples 8 to 12 below describes results obtained using the degummingassay of this example.

Example 8: Enzyme Robustness to Different Acidity of the Pretreatmentsof the Oil

The PI-specific phospholipase C of SEQ ID NO: 3 was applied in thedegumming assay testing different acid/base pretreatments of the crudeoil 3. The diglyceride content after enzymatic degumming at 50° C. and60° C. for 1 and 3 hours, were measured. The results are shown in inTable 13.

TABLE 13 Increase of diglyceride after enzyme treatment of acid/basepretreated soybean oil measured by HPLC-ELSD. Acid/base pretreatment ofoil 50 C. 60 C. Enz. Reaction time (hours) 1 3 1 3 0.05% PA/0.5 eqv NaOH0.19 0.37 0.37 0.37 0.05% PA/1.0 eqv NaOH 0.06 n.d. 0.38 0.41 0.05%PA/1.5 eqv NaOH 0.29 0.42 0.37 0.42 no acid/base 0.30 0.32 0.49 0.43

In the degumming assay the PI-specific phospholipase C of SEQ ID NO: 3converts PI-phospholipids into diglycerides in a few hours. Fullconversion is achieved after 3 hours based on the assumption that the182 ppm P originating from PI (measured by LCMS) is equal to 0.50%PI-phospholipid (Mw PI˜857 g/mol, Mw P˜31 g/mol) equal to 0.40% DGincrease (80% of phospholipid molecule). The enzyme showed goodperformance in water degumming (no acid/base) as well as acid assisteddegumming followed by base neutralization with varying concentrations ofNaOH. This demonstrated robustness towards varying pH conditions in thedegumming process.

Example 9: Effect of Enzyme Dosage

The PI-specific phospholipase C of SEQ ID NO: 3 was applied in thedegumming assay at various enzyme dosages in the crude oil 3. Thediglyceride content after enzymatic degumming at 60° C. for 1, 2, 3 and5 hours, were measured (oil pretreated with 0.05% phosphoric acid/1.5eqv. NaOH). The results are shown in table 14.

TABLE 14 Increase of diglyceride after enzyme treatment of acid/basepretreated soybean oil measured by HPLC-ELSD. DG increase after x hoursenz. Reaction/ Enzyme dosing mg EP/kg oil 1 2 3 5 1 0.19 0.26 0.31 0.382 0.32 0.38 0.32 0.43 4 0.31 0.38 0.33 0.51 6 0.38 0.40 0.44 0.52 200.48 0.59 0.54 0.52

In the degumming assay the PI-specific phospholipase C of SEQ ID NO: 3converted PI-phospholipids into diglycerides in 1-5 hours at enzymedosage from 1 to 20 mg EP/kg oil. Data demonstrates a dose-responseeffect with faster phospholipid conversion to diglyceride at higherenzyme dosing. PI content after degumming was reduced to less than 1mg/kg oil in all cases (measured by LCMS) and shows that the enzymeattacks the PI species.

Example 10: Combination of PI-Specific PLC and PC, PE-Specific PLC at50° C.

The PI-specific phospholipase C of SEQ ID NO: 3 was applied in thedegumming assay at 50° C. in combination with the PC, PE-specificphopholipase Purifine PLC and the PLC of SEQ ID NO: 22 applying crudeoil 3 pretreated with 0.05% phosphoric acid/1.5 eqv. NaOH. Thediglyceride content after enzymatic degumming for 1, 2, 3 and 5 hours,were measured as well as the metal content. The results are shown inTables 15A+B.

TABLE 15A Increase of diglyceride after enzyme treatment of 0.05%phosphoric acid/1.5 eqv. NaOH pretreated soybean oil measured byHPLC-ELSD. Enz. Dosage Enz. Reaction time (hours) ENZ (mg EP/kg oil) 1 23 5 PurifinePLC 4 0.23 0.30 0.36 0.43 SEQ ID NO: 3 4 0.09 0.06 0.08 0.07SEQ ID NO: 22 4 0.04 0.12 0.06 0.20 SEQ ID NO: 3 + 4 + 4 0.19 0.27 0.320.47 SEQ ID NO: 22 SEQ ID NO: 3 + + 4 + 4 0.45 0.59 0.69 0.68 PurifineSEQ ID NO: 3 +  4 + 10 0.29 0.44 0.61 0.80 SEQ ID NO: 22

TABLE 15B Ca, Mg, P composition (mg/kg oil) measured by ICP-OES after 5hours enzyme treatment. Enzyme Dosage (mg EP/kg oil) Ca Mg P Crude oil 371 74 998 Purifine 4 7 5 5.3 SEQ ID NO: 3 4 5 5 5.5 SEQ ID NO: 22 4 6 53.8 SEQ ID NO: 3 + 4 + 4 6 5 7 SEQ ID NO: 22 SEQ ID NO: 3 +  4 + 10 5 54.3 SEQ ID NO: 22 SEQ ID NO: 3 + 4 + 4 6 5 4.7 Purifine

Degumming with the PI-specific phospholipase C of SEQ ID NO: 3 combinedwith PC, PE specific PLC (purifine PLC or SEQ ID NO: 22) resulted insignificant diglyceride formation at 50° C. The blends resulted in acombined effect compared to the individual solutions seen by a greaterDG increase. Phosphorus content in degummed oil was reduced to adesirable end level below 5 mg/kg and is equal to ‘full’ conversion.

Example 11 PI-Specific PLC in Combination with PC, PE Specific PLC at60° C.

The PI-specific phospholipase C of SEQ ID NO: 3 was applied in thedegumming assay at 60° C. alone or in combination with the PC,PE-specific PLC of SEQ ID NO: 22 in crude oil 3 pretreated with 0.05%phosphoric acid/1.5 eqv. NaOH. The diglyceride content after enzymaticdegumming for 1, 2, 3 and 5 hours, were measured. The results are shownin table 16.

TABLE 16 Increase of diglyceride after enzyme treatment of 0.05%phosphoric acid/1.5 eqv. NaOH pretreated crude soybean oil measured byHPLC-ELSD. Enz. Dosage Enz. Reaction time (hours) ENZ (mg EP/kg oil) 1 23 5 SEQ ID NO: 3 4 0.22 0.29 0.29 0.35 SEQ ID NO: 22 4 0.20 0.40 0.470.82 SEQ ID NO: 3 + 4 + 4  0.48 0.84 0.96 1.18 SEQ ID NO: 22 SEQ ID NO:3 + 4 + 10 0.66 0.93 1.09 1.21 SEQ ID NO: 22

Degumming with the PI-specific phospholipase C of SEQ ID NO: 3 combinedwith the PC, PE-specific PLC of SEQ ID NO: 22 at 60° C. resulted in acombined effect compared to the effect of the individual enzymes, andconverted major parts of phospholipids (up to 87% at conditions tested(60° C., 5 hours). Calculation is based on the assumption that 864 ppm Ptotal measured by LC/MS is equal to 2.15 wt % phospholipid (AverageMw˜772 g/mol) equal to max 1.72% DG increase obtainable (80% ofphospholipid molecule). LCMS results shows less than 4 ppm P from PI,PC, PE in degummed oil sample and confirms that the blend attack PC; PE;PI phospholipid species.

Example 12: PI-Specific PLC in Combination with PC, PE Specific PLC at60° C.

The PI-specific phospholipase C of SEQ ID NO: 3 was applied in thedegumming assay at 60° C. alone and in combination with Purifine PLC orSEQ ID NO: 20 in crude oil 2 pretreated with 0.05% phosphoric acid/1.5eqv. NaOH. The diglyceride content after enzymatic degumming for 3 and 5hours was measured. The results are shown in Table 17.

TABLE 17 Increase of diglyceride after enzyme treatment of 0.05%phosphoric acid/1.5 eqv. NaOH pretreated crude soybean oil measured byHPLC-ELSD. Enz. Dosage Enz. Reaction time (hours) ENZ (mg EP/kg oil) 3 h5 h SEQ ID NO: 3 4 0.16 0.17 PurifinePLC 4 0.62 0.65 SEQ ID NO: 20 40.15 0.27 SEQ ID NO: 20 10  0.32 0.48 SEQ ID NO: 3 + 4 + 4 0.67 0.75Purifine SEQ ID NO: 3 + 4 + 4 0.30 0.43 SEQ ID NO: 20 SEQ ID NO: 3 + 4 + 10 0.48 0.63 SEQ ID NO: 20

The phospholipid species PI, PE and PC which are the species hydrolyzedby the applied enzymes make up 60% of the total 732 ppm phospholipid inthe used crude oil determined by LC/MS, equaling to max 0.86% DGincrease upon full hydrolysis. Degumming with the PI-specificphospholipase C of SEQ ID NO: 3 combined with the PC, PE-specific PLC ofSEQ ID NO: 20 or Purifine PLC results in a combined effect compared tothe effect of the individual enzymes and converts up to 51% of the totalphospholipids at 60° C., 5 hours. This corresponds to hydrolysis ofmajority of the PI, PE and PC in the crude oil sample. Calculations arebased on phospholipid average MW 772 g/mol.

Example 13: Degumming Using PI-Specific PLC in Combination with PC, PESpecific PLC at 60° C.

The PC, PE-specific phospholipase C (SEQ ID NO: 22 (amino acids25-283)), SEQ ID NO: 25 and SEQ ID NO:27 was applied in the degummingassay at 60° C. alone and in combination with PI-specific phospholipaseC of SEQ ID NO: 3 in crude oil 5. The phospholipases were dosed asvolume filtered fermentation broth (2 or 4 ml) per 50 g crude oil apartfrom Purifine dosed as 4 mg enzyme protein per kg oil. The diglycerideincrease after enzymatic degumming for 2 and 5 hours are shown in table18. The water degummed oil was post-treated with 0.09% phosphoric/0.5eqv. NaOH before the phosphorous content was measured by ICP-method.

TABLE 18 Increase of diglyceride after enzyme treatment measured byHPLC- Corona Veo and phosphorous content measured by ICP after oilpost-treatment with of 0.09% phosphoric acid/0.5 eqv. NaOH. Total PEnzyme DG increase (wt %) by ICP dosing as function of (mg/kg per 50SPECI- reaction time (hours) oil) ENZ g oil FICITY 2 5 5 h Purifine 4 mgEP/ PC, PE 0.19 0.13 14 kg oil SEQ ID 22 2 ml PC, PE 0.37 0.27 13 (a.a.25-283) Seq ID NO: 25 2 ml PC, PE 0.11 0.10 16 SEQ ID 27 2 ml PC, PE0.14 0.21 22 SEQ ID NO: 2 2 + 4 ml PI; PC; PE 0.18 0.60 21 (a.a. 25-283)& 3 and 22 SEQ ID NO: 2 2 + 2 ml PI; PC; PE 0.16 0.60 18 (a.a. 25-283) &3 and 24&25 SEQ ID NO: 2 2 + 2 ml PI; PC; PE 0.22 0.66 30 (a.a. 25-283)& 3 and 27

The phospholipid species PI, PE and PC which are the species hydrolyzedby the applied enzymes make up 81% of the total 583 ppm phospholipid inthe used crude oil determined by LC/MS, equaling to max 0.95% DGincrease upon full hydrolysis. Calculations are based on conversionfactor from phosphorous to phospholipids of 0.0025 and that diglyceridesconstitute 80% of the phospholipid molecule. Degumming with thePI-specific phospholipase C of SEQ ID NO: 3 combined with the PC,PE-specific PLC of SEQ ID NO: 22 (amino adds 25-283), SEQ ID NO: 25 orSEQ ID NO: 27 results in a combined effect compared to the effect of theindividual enzymes and converts up to 70% of the total accessiblephospholipids at 60° C., 5 hours.

Example 14: Degumming Using PI-Specific PLC in Combination with PC, PESpecific PLC at 60° C.

The PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino adds 25-283)was applied in the degumming assay at 60° C. alone and in combinationwith PI-specific phospholipase C (SEQ ID NO: 9 and SEQ ID NO: 15) usingcrude oil 5. The PI-specific phospholipase Cs were dosed as volumefiltered fermentation broth (2 ml) per 50 g crude oil while the PC;PE-specific phospholipase Cs were dosed as purified mg enzyme proteinper kg oil (4 mg EP/kg oil). The diglyceride increase after enzymaticdegumming for 2 and 5 hours are shown in table 19. The water degummedoil was post-treated with 0.09% phosphoric/0.5 eqv. NaOH before thephosphorous content was measured by ICP-method.

TABLE 19 Increase of diglyceride after enzyme treatment measured byHPLC- Corona Veo and phosphorous content measured by ICP after oilpost-treatment with of 0.09% phosphoric acid/0.5 eqv. NaOH. Total P DGincrease (wt %) by ICP Dosing as function of (mg/kg per 50 SPECI-reaction time (hours) oil) Enzyme g oil FICITY 2 5 5 h Blank 0.23 0.1216 Purifine 4 mg EP PC, PE 0.56 0.48 16 SEQ ID NO: 22 4 mg EP PC; PE0.54 0.43 16 (a.a. 25-283) SEQ ID NO: 9 2 ml PI 0.39 0.22 13 SEQ ID NO:15 2 ml PI 0.40 0.27 17 SEQ ID NO: 22 4 mg EP + PC; PE; 0.66 0.55 13(a.a. 25-283) + 15 2 ml PI SEQ ID NO: 22 4 mg EP + PC; PE; 0.47 0.51 12(a.a. 25-283) + 9 2 ml PI

The phospholipid species PI, PE and PC which are the species hydrolyzedby the applied enzymes make up 81% of the total 583 ppm phospholipid inthe used crude oil determined by LC/MS, equaling to max 0.95% DGincrease upon full hydrolysis. Degumming with the PC; PE-specificphospholipase C of SEQ ID NO: 22 (amino adds 25-283) combined with thePI-specific PLC of SEQ ID NO: 9 and 15 results in a combined effectcompared to the effect of the individual enzymes and converts up to 70%of the total accessible phospholipids at 60° C., 2 hours.

Example 15: Water Degumming Using PI-Specific PLC in Combination withPC, PE Specific PLC at 60° C.

The PI-specific phospholipase C of SEQ ID NO: 3 was applied in thedegumming assay at 60° C. alone and in combination with PC, PE-specificphospholipase C (SEQ ID NO: 22 (amino adds 25-283), SEQ ID NO: 25 andSEQ ID NO: 27 (amino adds 25-283)) in crude oil 4. Purifine and SEQ IDNO: 22 (amino adds 25-283) were dosed as mg purified enzyme protein perkg oil while the rest were dosed as volume filtered fermentation brothper 40 g crude oil. The diglyceride increase after enzymatic waterdegumming (no acid/base oil treatment) for 2 and 5 hours as well as andphosphorous content measured by ICP are shown in Table 20.

TABLE 20 Increase of diglyceride after enzyme treatment measured byHPLC-Corona Veo and phosphorous content measured by ICP. Total P DGincrease (wt %) by ICP as function of (mg/kg Enzyme SPECI- reaction time(hours) oil) Enzyme Dosing FICITY 2 5 5 hours Purifine 4 mg EP/ PC; PE0.96 0.88 1.4 kg oil SEQ ID NO: 22 4 mg EP/ PC; PE 0.82 0.89 1.6 (a.a.25-283) kg oil SEQ ID NO: 25 1.6 ml/ PC; PE 0.48 0.75 1.5 40 g oil SEQID NO: 27 1.6 ml/ PC; PE 0.52 0.88 0.7 (a.a. 25-283) 40 g oil SEQ ID NO:3 1.6 ml/ PI 0.25 0.27 0.8 40 g oil SEQ ID NO: 3 + 1.6 ml + PC; PE; 1.001.18 1.3 22 (a.a. 24-283) 4 mg EP PI SEQ ID NO: 3 + 1.6 ml + PC; PE;0.91 1.16 1.3 25 1.6 ml PI SEQ ID NO: 3 + 1.6 ml + PC; PE; 0.93 1.24 1.327 (a.a. 25-283) 1.6 ml PI

Degumming with the PI-specific phospholipase C of SEQ ID NO: 3 combinedwith PC, PE specific PLC (SEQ ID NO: 22 (amino acids 25-283), SEQ ID NO:25 and SEQ ID NO: 27 (amino adds 25-283)) resulted in significantdiglyceride formation. The phospholipid species PI, PE and PC which arethe species hydrolyzed by the applied enzymes make up 98% of the total974 ppm phospholipid in the used crude oil determined by LC/MS, equalingto max 1.91% DG increase upon full hydrolysis. Calculations are based onconversion factor from phosphorous to phospholipids of 0.0025 and thatdiglycerides constitute 80% of the phospholipid molecule. Degumming withthe PI-specific phospholipase C of SEQ ID NO: 3 combined with the PC,PE-specific PLC of SEQ ID NO: 27 results in a combined effect comparedto the effect of the individual enzymes and converts up to 65% of thetotal accessible phospholipids at 60° C., 5 hours. Phosphorus content indegummed oil was reduced to below 5 mg/kg.

Example 16: Water Degumming Using PI-Specific PLC in Combination withPC, PE Specific PLC at 60° C.

The PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino adds 25-283)was applied in the degumming assay at 60° C. alone and in combinationwith PI-specific phospholipase C (SEQ ID NO: 9 and SEQ ID NO: 15) usingcrude oil 4. The PI-specific phospholipase Cs were dosed as 2 mlfiltered fermentation broth per 50 g crude oil while the PC; PE-specificphospholipase Cs were dosed as 4 mg enzyme protein per kg oil. Thediglyceride increase after enzymatic water degumming (no acid/base oiltreatment) for 2 and 5 hours as well as and phosphorous content measuredby ICP are shown in Table 21.

TABLE 21 Increase of diglyceride after enzyme treatment measured byHPLC-Corona Veo and phosphorous content measured by ICP. Total P DGincrease (wt %) by ICP as function of (mg/kg SPECI- reaction time(hours) oil) Enzyme FICITY 2 5 5 hours Blank 0.05 0.04 5.3 Purifine 4 mgEP/ PC; PE 0.90 0.98 0.9 kg oil SEQ ID NO: 22 4 mg EP/ PC; PE 0.89 0.931.0 (a.a. 24-283) kg oil SEQ ID NO: 9 2 ml/ PI 0.27 0.33 1.2 50 g oilSEQ ID NO: 15 2 ml/ PI 0.30 0.35 2.1 50 g oil SEQ ID NO: 22 4 mg EP +PC; PE; 1.18 1.28 1.3 (a.a. 25-283) + 15 2 ml PI SEQ ID NO: 22 4 mg EP +PC; PE; 1.15 1.32 1.5 (a.a. 25-283) + 9 2 ml PI

Degumming with the PC, PE-specific phospholipase C of SEQ ID NO: 22(amino adds 25-283) combined with PI-specific phospholipase C of SEQ IDNOS: 9 and 15 resulted in significant diglyceride formation. Thephospholipid species PI, PE and PC which are the species hydrolyzed bythe applied enzymes make up 98% of the total 974 ppm phospholipid in theused crude oil determined by LC/MS, equaling to max 1.91% DG increaseupon full hydrolysis. Degumming with the with the PC, PE-specific PLC ofSEQ ID NO: 22 (amino adds 25-283) combined with PI-specificphospholipase C of SEQ ID NO: 9 and 15 combined results in a combinedeffect compared to the effect of the individual enzymes and converts upto 69% of the total accessible phospholipids at 60° C., 5 hours.Phosphorus content in degummed oil was reduced to below 5 mg/kg.

Example 17: Water Degumming Using PI-Specific PLC in Combination withPC, PE Specific PLC at 60° C.

The PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids.25-283) was applied in the degumming assay at 60° C. alone and incombination with PI-specific phospholipase C (SEQ ID NO: 3 and SEQ IDNO: 6) using crude oil 4. The phospholipases were dosed as 4 ml filteredfermentation broth per 50 g crude oil. The diglyceride increase afterenzymatic water degumming (no acid/base oil treatment) for 2 and 5 hoursas well as the phosphorous content measured by ICP after 5 hours areshown in Table 22.

TABLE 22 Increase of diglyceride after enzyme treatment measured byHPLC-Corona Veo and phosphorous content measured by ICP. Total P DGincrease (wt %) by ICP Dosing as function of (mg/kg per 50 SPECI-reaction time (hours) oil) Enzyme g oil FICITY 2 5 (5 h) SEQ ID NO: 22 4ml PC, PE 0.72 0.93 0.8 (a.a. 25-283) SEQ ID NO: 23 + 4 + 4 ml PC; PE;0.93 1.24 1.7 22 (a.a. 25-283) PI SEQ ID NO: 6 + 4 + 4 ml PC; PE; 0.961.24 1 22 (a.a. 25-283) PI

Degumming with the PC, PE-specific phospholipase C of SEQ ID NO: 22(amino adds 25-283) combined with PI-specific phospholipase C of SEQ IDNOS: 3 and 6 resulted in combined effect compared to the effect of theindividual enzymes and converts up to 65% of the total accessible PI;PC; PE phospholipids at 60° C., 5 hours. Phosphorus content in degummedoil was reduced to a desirable end level below 5 mg/kg equal to ‘full’ Premoval/reduction.

Example 18: Degumming Using PI-Specific PLC (SEQ ID NO: 38 Amino Acids25-280) in Degumming Assay at 60° C.

The PC, PE-specific phospholipase C of SEQ ID NO: 38 (amino acids25-280) was applied in the degumming assay at 60° C. using crude oil 5(diff oil). The crude oil 5 was pre-treated with 0.09% phosphoric acidand 1.5 molar equivalents of NaOH prior to incubation with enzymes. Thephospholipase was dosed as 30 mg enzyme protein per kg oil. Thediglyceride increase after enzymatic degumming for 2, 4, 6 and 24 hourswas measured by HPLC-Corona Veo as well as the phosphorous contentmeasured by ICP after 24 hours are shown in Table 23.

TABLE 23 Increase of diglyceride after enzyme treatment measured byHPLC-Corona Veo and phosphorous content measured by ICP. Total P by ICPas function of reaction DG increase (wt %) as function of time (hours)Enz dosing reaction time (hours) (mg/kg oil) Enzyme mg EP/kg oil 2 4 624 2 24 Blank 0.08 0.07 0.06 0.14 34 32 SEQ ID NO: 30 0.43 0.43 0.410.60 40 39 38 (a.a. 25- 280)

The phospholipid species PC and PE which are the species hydrolyzed bythe applied enzymes make up 70% of the total 583 ppm phospholipid in theused crude oil determined by LC/MS, equaling to max 0.82% DG increaseupon full hydrolysis. Calculations are based on conversion factor fromphosphorous to phospholipids of 0.0025 and that diglycerides constitute80% of the phospholipid molecule. Degumming with the phospholipase C ofSEQ ID NO: 38 (amino acids 25-280) converts up to 53% of the totalaccessible phospholipids at 60° C., 2 hours.

Example 19: SEQ ID NO: 22 (Amino Acids 39-283) and SEQ ID NO: 38 (AminoAcids 36-280) Tested in Degumming Assay at 60° C. Citric Acid/BasePretreatment

The PC, PE-specific phospholipase C of SEQ ID NO: 22 (amino acids39-283) and the PC, PE-specific phospholipase C of SEQ ID NO: 38 (aminoacids 36-280) were applied in the degumming assay at 60° C. using crudeoil 5 (diff oil). The crude oil 5 was pre-treated with 0.065% citricacid and 1.5 molar equivalents of NaOH prior to incubation with enzymes.The phospholipase was dosed as 4 ml filtered fermentation broth per 50 gcrude oil. The diglyceride increase after enzymatic degumming for 2 and5 hours as well as the phosphorous content measured by ICP after 2 hoursare shown in Table 24.

TABLE 24 Increase of diglyceride after enzyme treatment measured byHPLC-Corona Veo and phosphorous content measured by ICP. DG increase (wt%) Total P by Enz dosing as function of ICP (2 h per 50 reaction time(hours) WDG) Enz/SEQ ID NO: g oil 2 5 2 hours Blank 4 ml 0.02 0.04 12SEQ ID NO: 38 4 ml 0.50 0.63 12 (a.a. 36-280) SEQ ID NO: 22 4 ml 0.460.49 15 (a.a. 39-283)

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A method for reducing the content of phospholipids in an oilcomposition, the method comprising a) providing an oil compositioncontaining a quantity of phospholipids, b) contacting said oilcomposition with a phosphatidylinositol phospholipase C and a PC andPE-specific phospholipase C under conditions sufficient for the enzymesto react with the phospholipids to create diglyceride and phosphateester; and c) separating the phosphate ester from the oil composition.2. The method of claim 1, wherein said phosphatidylinositolphospholipase C is from the genus of Pseudomonas.
 3. The method of claim1, wherein the oil is an edible oil.
 4. The method of claim 1, whereinthe oil is selected from crude oil, water degummed oil, caustic refinedoil and acid degummed oil.
 5. The method of claim 1, wherein the oilcomprises phosphatidylcholine (PC), phosphatidylethanolamine (PE) andphosphatidyl inositol (PI).
 6. The method of claim 5, wherein the oilcomprises at least 50 ppm phosphorus originating from phosphatidylinositol (PI). 7-11. (canceled)
 12. A polypeptide havingphosphatidylinositol phospholipase C activity, selected from the groupconsisting of: a) a polypeptide having at least 91% sequence identity tothe mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by apolynucleotide that hybridizes under medium stringency conditions withi) the mature polypeptide coding sequence of SEQ ID NO: 1, or ii) thefull-length complement of (i); c) a polypeptide encoded by apolynucleotide having at least 90% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1; d) a variant of the maturepolypeptide of SEQ ID NO: 2 comprising a substitution, deletion, and/orinsertion at one or more positions; and e) a fragment of the polypeptideof (a), (b), (c), or (d) that has phosphatidylinositol phospholipase Cactivity. 13-20. (canceled)
 21. A polypeptide having PC and PE specificphospholipase C activity, selected from the group consisting of: a) apolypeptide having at least 70% sequence identity to the maturepolypeptide of SEQ ID NO: 19; b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with i)the mature polypeptide coding sequence of SEQ ID NO: 18, or ii) thefull-length complement of (i); c) a polypeptide encoded by apolynucleotide having at least 70% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 18; d) a variant of the maturepolypeptide of SEQ ID NO: 19 comprising a substitution, deletion, and/orinsertion at one or more positions; and e) a fragment of the polypeptideof (a), (b), (c), or (d) that has PC and PE specific phospholipase Cactivity. 22-29. (canceled)
 30. A polynucleotide encoding a polypeptideof claim
 12. 31. A nucleic acid construct or expression vectorcomprising the polynucleotide of claim 30 operably linked to one or morecontrol sequences that direct the production of the polypeptide in anexpression host.
 32. A recombinant host cell comprising thepolynucleotide of claim 30 operably linked to one or more controlsequences that direct the production of the polypeptide. 33-35.(canceled)
 36. A method of producing a polypeptide havingphosphatidylinositol phospholipase C activity or PC and PE specificphospholipase C activity, comprising cultivating the host cell of claim32 under conditions conducive for production of the polypeptide.
 37. Themethod of claim 36, further comprising recovering the polypeptide.
 38. Acomposition comprising the polypeptide of claim
 12. 39. A compositioncomprising the polypeptide of claim
 21. 40. (canceled)
 41. Thecomposition of claim 38, wherein the phosphatidylinositol phospholipaseC is: a) a polypeptide comprising an amino acid sequence selected fromthe group consisting of: i) amino acid residues 26-322 of SEQ ID NO: 2or amino acid residues 1-298 of SEQ ID NO: 3; ii) amino acid residues26-323 of SEQ ID NO: 5 or amino acid residues 1-299 of SEQ ID NO: 6;iii) amino acid residues 26-323 of SEQ ID NO: 8 or amino acid residues1-299 of SEQ ID NO: 9; iv) amino acid residues 26-323 of SEQ ID NO: 11or amino acid residues 1-296 of SEQ ID NO: 12; v) amino acid residues26-322 of SEQ ID NO: 14 or amino acid residues 1-298 of SEQ ID NO: 15;vi) amino acid residues 26-322 of SEQ ID NO: 17; and vii) amino acidresidues 28-339 of SEQ ID NO: 35; or b) a polypeptide comprising anamino acid sequence which has at least 75% identity to one of the aminoacid sequences in a); or c) a functional fragment of a) or b). 42.(canceled)
 43. The composition of claim 39, wherein the PC andPE-specific phospholipase C polypeptide is: a) a polypeptide comprisingan amino acid sequence selected from the group consisting of: i) aminoacid residues 34-278 of SEQ ID NO: 19 or amino acid residues 1-246 ofSEQ ID NO: 20; ii) amino acid residues 25-283 of SEQ ID NO: 22 or aminoacid residues 39-283 of SEQ ID NO: 22; iii) amino acid residues 25-283of SEQ ID NO: 24 or amino acid residues 39-283 of SEQ ID NO: 24 or aminoacid residues 1-260 of SEQ ID NO: 25; iv) amino acid residues 39-283 ofSEQ ID NO: 27; v) amino acid residues 52-289 of SEQ ID NO: 29 or aminoacid residues 1-263 of SEQ ID NO: 30; vi) amino acid residues 21-282 ofSEQ ID NO: 32 or amino acid residues 38-282 of SEQ ID NO: 32; vii) aminoacid residues 25-280 of SEQ ID NO: 38 or amino acid residues 36-280 ofSEQ ID NO: 38; and viii) Purafine; or b) a polypeptide comprising anamino acid sequence which has at least 75% identity to one of the aminoacid sequences in a); or c) a functional fragment of a) or b).